CN112105634A

CN112105634A - Novel interleukin-2 and uses thereof

Info

Publication number: CN112105634A
Application number: CN201980029372.8A
Authority: CN
Inventors: 康立山; 付凤根; 周帅祥; 史新震; 刘军建
Original assignee: Innovent Biologics Suzhou Co Ltd
Current assignee: Innovent Biologics Suzhou Co Ltd
Priority date: 2018-09-21
Filing date: 2019-09-20
Publication date: 2020-12-18
Anticipated expiration: 2039-09-20
Also published as: TW202014432A; US20210213102A1; JP2022501009A; JP2023174651A; TWI801664B; CA3098765A1; CN112105634B; AU2019344875A1; WO2020057645A1; EP3854805A4; AU2019344875B2; EP3854805A1

Abstract

The present invention relates to novel interleukin 2(IL-2) muteins. The invention also provides fusion proteins, immunoconjugates comprising the IL-2 muteins, as well as nucleic acids encoding the IL-2 muteins, vectors and host cells comprising the nucleic acids. The invention further provides methods of making the IL-2 muteins, pharmaceutical compositions comprising the IL-2 muteins, and therapeutic uses of the muteins.

Description

Novel interleukin-2 and uses thereof

Technical Field

The present invention relates to novel interleukin 2(IL-2) muteins and uses thereof. In particular, the present invention relates to IL-2 muteins having improved properties, e.g., improved druggability, reduced IL-2R alpha receptor binding capacity and/or increased IL-2R beta receptor binding capacity, compared to wild-type IL-2. The invention also provides fusion proteins, immunoconjugates comprising the IL-2 muteins, as well as nucleic acids encoding the IL-2 muteins, vectors and host cells comprising the nucleic acids. The invention further provides methods of making the IL-2 muteins, pharmaceutical compositions comprising the IL-2 muteins, and therapeutic uses of the muteins.

Background

Interleukin-2 (IL-2), also known as T Cell Growth Factor (TCGF), is a protein composed primarily of activated T cells, particularly CD4⁺T helper cell produces pluripotent cytokines. In eukaryotic cells, human IL-2(uniprot: P60568) was synthesized as a precursor polypeptide of 153 amino acids, which, after removal of the N-terminal 20 amino acids, produced mature secreted IL-2. The sequences of IL-2 from other species have also been published, see NCBI Ref Seq No. NP032392 (mouse), NP446288 (rat) or NP517425 (chimpanzee).

Interleukin 2 has 4 antiparallel, amphipathic alpha helices, the 4 alpha helices forming the quaternary structure essential for its function (Smith, Science 240,1169-76 (1988); Bazan, Science257,410-413 (1992)). In most cases, IL-2 acts through three different receptors: interleukin 2 receptor alpha (IL-2R alpha; CD25), interleukin 2 receptor beta (IL-2R beta; CD122), and interleukin 2 receptor gamma (IL-2R gamma; CD 132). IL-2R β and IL-2R γ are critical for IL-2 signaling, while IL-2R α (CD25) is not essential for signaling, but can confer high affinity binding of IL-2 to the receptor (Krieg et al, Proc Natl Acad Sci 107,11906-11 (2010)). The trimeric receptor (IL-2. alpha. beta. gamma.) formed by the combination of IL-2R. alpha., beta, and gamma is the IL-2 high affinity receptor (KD about 10pM), the dimeric receptor (IL-2. beta. gamma.) composed of beta and gamma is the intermediate affinity receptor (KD about 1nM), and the IL-2 receptor formed by the alpha subunit alone is the low affinity receptor.

Immune cells express dimeric or trimeric IL-2 receptors. Dimeric receptor cytotoxic CD8⁺T cells and Natural Killer (NK) cells, while the trimer receptor is predominantly on activated lymphocytes and CD4⁺CD25 ⁺FoxP3 ⁺Expression on suppressive regulatory T cells (tregs) (Byman, o. and span.j.nat. rev. immunol.12,180-190 (2012)). Since effector T cells and NK cells in a resting state do not have CD25 on the cell surface, they are relatively insensitive to IL-2. Whereas Treg cells consistently express the highest level of CD25 in vivo, IL-2 normally preferentially stimulates Treg cell proliferation.

IL-2 mediates multiple effects in immune responses by binding to IL-2 receptors on different cells. In one aspect, as an immune system stimulator, IL-2 can stimulate T cell proliferation and differentiation, induce Cytotoxic T Lymphocyte (CTL) production, promote B cell proliferation and differentiation and immunoglobulin synthesis, and stimulate the production, proliferation and activation of Natural Killer (NK) cells, and thus has been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infections. On the other hand, IL-2 can promote immunosuppressive CD4⁺CD25 ⁺Maintenance of regulatory T cells (i.e., Treg cells) (Fontent et al, Nature Immunol6,1142-51 (2005); D' Cruz and Klein, Nature Immunol6,1152-59 (2005); Maloy and Powrie, Nature Immunol6,1171-72(2005)) and mediate activation-induced cell death(AICD) and in the establishment and maintenance of immune tolerance against self-antigens and tumor antigens (Lenardo et al, Nature 353: 858(1991)), leading to tumor tolerance by AICD and immunosuppression by activated Treg cells in patients. In addition, high doses of IL-2 can cause Vascular Leak Syndrome (VLS) in patients. IL-2 has been shown to induce pulmonary edema by direct binding to the IL-2 trimer receptor (IL-2. alpha. beta. gamma.) on lung endothelial cells (Krieg et al, Proc Nat Acad Sci USA107,11906-11 (2010)).

To overcome the above-mentioned problems associated with IL-2 immunotherapy, it has been proposed to reduce the toxicity and/or improve the efficacy of IL-2 therapy by altering the selectivity or preference of IL-2 for different receptors. For example, it has been proposed to use complexes of IL-2 monoclonal antibodies with IL-2 to induce CD122 by targeting IL-2 to cells expressing CD122 but not CD25^highPreferential expansion of the population enhances the therapeutic effect of IL-2 in vivo (Boyman et al, Science 311,1924-1927 (2006)). Oliver AST et al (US2018/0142037) proposed the introduction of triple mutations F42A/Y45A/L72G at amino

acid residue positions

42,45 and 72 of IL-2 to reduce the affinity for the IL-2R α receptor. Aron M.Levin et al (Nature, Vol 484, p529-533, DOI 10.1038/Nature10975) proposed an IL-2 mutant, IL-2, called "superakine"^H9The mutant contains a quintuple mutation L80F/R81D/L85V/I86V/I92F and has enhanced IL-2R beta binding, so that the binding to CD25 is improved^-Stimulation of the cells, but still high binding to CD25 was maintained. Rodrigo Vazquez-Lombardi et al (Nature Communications,8:15373, DOI:10.1038/ncomms15373) proposed a triple mutant human IL-2 mutein IL-2^3XThe protein has residue mutations R38D-K43E-E61R at amino acid residue positions 38,43 and 61, respectively, resulting in the mutein not binding to IL-2R α, but the mutein activating CD25^-Weak effect on cells, CD25⁺The activation bias of the cells still exists. Furthermore, Rodrigo Vazzez-Lombardi et al also proposed to improve the pharmacodynamic properties of interleukins by preparing interleukin 2-Fc fusions, but the fusion proteins were expressed in low amounts and were prone to aggregate formation.

In view of the role of IL-2 in immune regulation and disease, there remains a need in the art to develop new IL-2 molecules with improved properties, particularly IL-2 molecules that exhibit advantageous production, purification, and improved pharmacodynamic properties.

Summary of The Invention

The present invention meets the above-described needs by providing novel IL-2 muteins having improved druggability and/or improved IL-2 receptor selectivity/bias relative to wild-type IL-2.

Thus, in one aspect, the invention provides novel IL-2 muteins. In some embodiments, the IL-2 muteins of the present invention have one or more of the following properties:

(i) improved druggability, in particular improved expression and/or purification when expressed in mammalian cells,

(ii) reducing or eliminating binding to IL-2R alpha;

(iii) enhancing binding to IL-2R beta.

In some embodiments, the invention provides IL-2 muteins comprising an introduced mutated glycosylation motif at the binding interface of IL-2 and IL-2R α; in other embodiments, the invention provides IL-2 muteins comprising a deletion and/or substitution in the B 'C' loop region of IL-2 to have a shortened loop sequence; in still other embodiments, the invention provides IL-2 muteins having both a mutated glycosylation motif and a shortened B 'C' loop sequence.

In addition, the invention provides fusion proteins and immunoconjugates comprising the IL-2 mutein, pharmaceutical compositions and combination products; nucleic acids encoding IL-2 muteins, vectors and host cells comprising the same; and methods of producing the IL-muteins, fusion proteins and immunoconjugates of the invention.

Furthermore, the invention also provides methods of treating diseases and methods and uses of stimulating the immune system of a subject using the IL-2 muteins and fusions and immunoconjugates of the invention. In some embodiments, the methods of the invention result in CD25 in a subject^-Strong activation and expansion of effector T cells and NK cells. In the process ofIn some embodiments, the IL-2 mediated immune down-regulation on Treg cells can be effectively reduced by the methods of the invention.

The invention is further illustrated in the following figures and detailed description. However, these drawings and specific embodiments should not be construed as limiting the scope of the invention, and modifications readily ascertainable by those skilled in the art would be included within the spirit of the invention and the scope of the appended claims.

Drawings

FIG. 1 shows the crystal structures of IL-2 and IL-2R α (PDB:1Z92) (A) and a schematic diagram of the structure of IL-2 glycosylation modified proteins (B).

FIG. 2 shows the crystal structure of IL-2 (PBD:2ERJ) (A) and the B ' C ' loop structure of IL-2 and human IL15 in human and murine (B ') superpose (B).

FIG. 3 shows the HPLC purity profile of a sample after IL-2R α purification.

FIG. 4 shows the HPLC purity profile of a sample after IL-2R β purification.

FIG. 5 shows some IL-2 selected and constructed^mutantFC at CD8⁺CD25 ^-/CD25 ⁺The signal curve for p-STAT5 activation on T cells.

FIG. 6 shows the mature protein sequence (SEQ ID NO:26) of human interleukin (IL-2) and its amino acid residue numbering, and shows exemplary IL-2 glycosylation mutants and IL-2 chimeric and truncated B 'C' loop mutants.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

The term "about," when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value.

The term "and/or" should be understood to mean any one of the options or a combination of any two or more of the options.

As used herein, the term "comprising" or "comprises" is intended to mean including the stated elements, integers or steps, but not excluding any other elements, integers or steps. When the term "comprising" or "includes" is used herein, unless otherwise specified, it also encompasses the presence of stated elements, integers or steps. For example, when referring to "contains" or "includes" a certain mutation or combination of mutations of IL-2 mutant protein, also intended to cover only the mutation or combination of mutations of IL-2 mutant protein.

In this context, wild-type "interleukin-2" or "IL-2" refers to a parent IL-2 protein, preferably a naturally occurring IL-2 protein, e.g., a native IL-2 protein derived from a human, mouse, rat, non-human primate, including both unprocessed (e.g., without removal of the signal peptide) and processed (e.g., with removal of the signal peptide) forms, as a template for introducing a mutation or combination of mutations of the invention. A full-length native human IL-2 sequence comprising a signal peptide is shown in SEQ ID NO. 29 and the sequence of its mature protein is shown in SEQ ID NO. 30. In addition, the expression also includes naturally occurring IL-2 allelic variants and splice variants, isoforms, homologs, and species homologs. The expression also includes natural IL-2 variants, for example, the variants can have at least 95% -99% or more with natural IL-2 or with no more than 1-10 or 1-5 amino acid mutations (especially conservative amino acid substitutions), and with natural IL-2 protein has substantially the same IL-2R alpha binding affinity and/or IL-2R beta binding affinity. Thus, in some embodiments, wild-type IL-2 may comprise an amino acid mutation that does not affect its binding to the IL-2 receptor as compared to the native IL-2 protein, e.g., the native human IL-2 protein with the mutation C125S introduced at position 125 (uniprot: P60568) is a wild-type IL-2 of the invention. An example of a wild-type human IL-2 protein comprising the C125S mutation is shown in SEQ ID NO 26. In some embodiments, the wild-type IL-2 sequence may have at least 85%, 95%, even at least 96%, 97%, 98%, or 99% or more amino acid sequence identity to the amino acid sequence of SEQ ID No. 26 or 29 or 30.

Herein, the amino acid mutation may be an amino acid substitution, deletion, insertion and addition. Any combination of substitutions, deletions, insertions and additions can be made to achieve the final mutein construct with the desired properties, such as reduced binding affinity for IL-2 ra. Amino acid deletions and insertions include deletions and insertions at the amino and/or carboxy terminus of the polypeptide sequence. For example, an alanine residue can be deleted at position 1 of full length human IL-2. In some embodiments, preferred amino acid mutations are amino acid substitutions. In other embodiments, the preferred amino acid mutation is an amino acid deletion. In some embodiments, the invention describes specific mutation in amino acid position introduced mutations to obtain with altered glycosylation motif IL-2 mutant protein. In some embodiments, the invention describes specific mutation in amino acid position introduced mutations to obtain with shortened B 'C' ring sequence of IL-2 mutant protein.

In the present invention, when referring to the amino acid position of the IL-2 protein, reference is made to the wild-type human IL-2 protein of SEQ ID NO:26 (also referred to as IL-2)^WT) The amino acid sequence (as shown in FIG. 6) was determined. Can be aligned by amino acid sequence alignment (e.g., using BLAST; available from http:// BLAST. ncbi. nlm. nih. gov/BLAST. cgi&PAGE_TYPE＝BlastSearch&LINK _ LOC ═ Basic Local Alignment Search Tool obtained from blastome, aligned using default parameters), the corresponding amino acid positions on other IL-2 proteins or polypeptides (including full-length sequences or truncated fragments) were identified. Thus, in the present invention, unless otherwise indicated, the amino acid position in the IL-2 protein or polypeptide is the amino acid position numbered according to SEQ ID NO: 26. For example, when referring to "F42", it is meant phenylalanine residue F at position 42 of SEQ ID NO 26, or an amino acid residue aligned at a corresponding position on other IL-2 polypeptide sequences.

In this paper, in reference to IL-2 mutant protein, according to the following manner describe the mutation. Amino acid substitutions are indicated as [ original amino acid residue/position/substituted amino acid residue ]. For example, a substitution of the amino acid at position 35 with asparagine (N) can be expressed as 35N, or K35N if the original amino acid residue at position 35 is lysine. When a substituted residue is denoted by X, e.g., 36X, it is meant that the amino acid at position 36 can be substituted with any residue, if X has the value of the particular residue, that position is substituted with the particular X residue defined. However, in the representation where only the original residues and positions are given, e.g. L36 and T37 in the mutated glycosylation motif K35N-L36-T37 of the invention, it means that no mutation occurs at said positions 36 and 37, i.e. the original residues L and T remain at positions 36 and 37.

Herein, the "percent sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window. Preferably, sequence identity is determined over the full length of the reference sequence (e.g., SEQ ID NO: 26). Methods of sequence alignment for comparison are well known in the art. Algorithms suitable for determining percent sequence identity include, for example, the BLAST and BLAST2.0 algorithms (see Altschul et al, Nuc. acids Res.25: 3389-.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological function of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Typical conservative amino acid substitutions are those in which one amino acid is substituted for another with similar chemical properties (e.g., charge or hydrophobicity). The following six groups each contain amino acids that can be typically conservatively substituted for each other: 1) alanine (a), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). For example, the wild-type IL-2 protein may have conservative amino acid substitutions relative to one of SEQ ID NOs 26,29 or 30, or only conservative amino acid substitutions. As another example, the mutant IL-2 proteins of the invention may have conservative amino acid substitutions, or only conservative amino acid substitutions, relative to the IL-2 mutein sequences specifically set forth herein (e.g., any one of SEQ ID NOs: 31-50).

"affinity" or "binding affinity" can be used to reflect the intrinsic binding capacity of the interaction between the members of a binding pair. The affinity of the molecule X for its binding partner Y can be determined by the equilibrium dissociation constant (K)_D) As used herein, the equilibrium dissociation constant is the dissociation and association rate constants (k, respectively)_disAnd k_on) The ratio of (a) to (b). Binding affinity can be measured by common methods known in the art. One particular method for measuring affinity is the Biofilm Layer Interference (BLI) technique assay herein.

Herein, an antibody binding molecule is a polypeptide molecule that can specifically bind to an antigen, e.g., an immunoglobulin molecule, an antibody or an antibody fragment, such as a Fab fragment and a scFv fragment.

Herein, an antibody Fc-fragment refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region, and may include native sequence Fc-fragments and variant Fc-fragments. In one embodiment, the human IgG heavy chain Fc fragment extends from Cys226 or from Pro230 of the heavy chain to the carboxy terminus. In another embodiment, the C-terminal lysine (Lys447) of the Fc-fragment may or may not be present. In other embodiments, the Fc fragment may comprise a mutation, for example, the L234A/L235A mutation. Unless otherwise indicated herein, the numbering of amino acid residues in an Fc fragment is according to the EU numbering system, also known as the EU index, as described in Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

Aspects of the invention are described in further detail in the following subsections.

1.IL-2 muteins of the present invention

The present invention provides in one aspect novel IL-2 muteins with improved druggability and/or improved IL-2 receptor selectivity/preference.

Advantageous biological Properties of the IL-2 muteins of the invention

IL-2 proteins trigger signaling and function by interacting with IL-2 receptors. Wild-type IL-2 shows different affinity for different IL-2 receptors. In resting effector cells (including CD 8)⁺Cytotoxic T cells and NK cells) express IL-2 β and γ receptors with lower affinity for wild-type IL-2. IL-2R alpha with high affinity to wild-type IL-2 is expressed on regulatory T cell (Treg) cells and activated effector cells. Due to high avidity, wild-type IL-2 will preferentially bind to IL-2ra on the cell surface, recruit IL-2R β γ, release downstream p-STAT5 signals through IL-2R β γ, stimulate Treg cells and activated effector cells. Thus, without being bound by theory, reducing or eliminating IL-2's affinity for IL-2R α receptor will reduce preferential activation of CD25 by IL-2⁺The cell tropism reduces the IL-2 mediated immune down-regulation effect of Treg cells. Without being bound by theory, maintaining or enhancing affinity for IL-2 beta receptors will retain or enhance IL-2 on effector cells such as CD8⁺Cytotoxic T cell and NK cell activation and thus IL-2 immune stimulation effect.

The present inventors have discovered that the expression and/or purity of IL-2 muteins, and/or the binding of IL-2 muteins to IL-2Ra can be improved, and/or reduced, by introducing one or more specific N-glycosylation motifs at the IL-2 and IL-2Ra receptor binding interface. Furthermore, the present inventors have also found that the expression and/or purity of IL-2 can be increased and at the same time the affinity for IL-2R β can be increased by replacing the B 'C' loop sequence of IL-2 itself with a short B 'C' loop sequence from another interleukin cytokine, such as IL-15, or by truncating the B 'C' loop sequence of IL-2 itself.

Thus, the present invention provides IL-2 muteins with improved properties. The IL-2 muteins of the present invention may have improved properties relative to wild-type IL-2, selected from, for example, one or more of the following: (i) improved expression and/or purity when expressed in mammalian cells; (ii) reduced or eliminated binding to the IL-2R α receptor; and/or (iii) enhanced binding to the IL-2R β receptor.

In some embodiments, the IL-2 muteins of the present invention have improved properties relative to wild-type IL-2, selected from, for example, one or more of the following:

(1) reduced or eliminated binding affinity to the IL-2R alpha receptor,

(2) enhanced binding affinity to the IL-2R β receptor;

(3) a reduced binding affinity to a high affinity IL-2R receptor (IL-2R α β γ);

(4) increased binding affinity to intermediate affinity IL-2R receptors (IL-2R β γ);

(5) in CD25⁺Cells (particularly activated CD 8)⁺T cells and Treg cells), in particular a reduced ability to activate STAT5 phosphorylation signal;

(6) resulting in reduced IL-2 mediated CD25⁺Cells (particularly activated CD 8)⁺T cells and Treg cells) activation and proliferation;

(7) reducing or eliminating the propensity of IL-2 to preferentially stimulate Treg cell proliferation;

(8) reducing the immune down-regulation effect of Treg cells under the induction of IL-2;

(9) retention or enhancement, especially enhancement, to CD25^-Cells (especially CD25)^-T effector cells and NK cells);

(10) results in increased IL-2 mediated effector T cell and NK cell activation and proliferation;

(11) results in increased immune stimulation;

(12) improving the anti-tumor effect.

In some embodiments, the IL-2 muteins of the present invention have the properties of (1) above, preferably further have one or more, especially all, properties selected from (3) and (5) to (8); more preferably still further has one or more, especially all, properties selected from (2) and (9) to (12). In some embodiments, the IL-2 muteins of the present invention have the properties of (2) above, preferably further have one or more, especially all, properties selected from (9) to (12); more preferably still further has one or more, especially all, properties selected from (1), (3) and (5) - (8).

In some preferred embodiments, the IL-2 muteins of the invention also have one or more of the following properties relative to wild-type IL-2: reduced in vivo toxicity mediated by binding of IL-2 to the high affinity receptor IL-2 α β γ.

In some embodiments, the IL-2 muteins of the present invention have improved pharmaceutical properties, e.g., one or more properties selected from the group consisting of: (i) the expression quantity of the IL-2 protein is better than that of the wild type IL-2 protein; (ii) stability superior to wild-type IL-2 protein; and (iii) easy purification to higher protein purity.

In some embodiments of the invention, the invention of the IL-2 mutant protein and wild type IL-2 compared with the expression level increase. In some embodiments of the invention, the increased expression occurs in a mammalian cell expression system. Expression levels can be determined by any suitable method that allows for quantitative or semi-quantitative analysis of the amount of recombinant IL-2 protein in cell culture supernatants, preferably after purification by one-step affinity chromatography. For example, the amount of recombinant IL-2 protein in a sample can be assessed by Western blotting or ELISA. In some embodiments, the IL-2 muteins of the present invention have an increased expression level in mammalian cells of at least 1.1-fold, or at least 1.5-fold, or at least 2-fold, 3-fold, or more than 4-fold, as compared to wild-type IL-2.

In some embodiments, the IL-2 mutein-Fc fusions of the present invention exhibit greater stability, e.g., less tendency to form aggregates, relative to wild-type IL-2 protein fusions, as shown by determining the purity of the purified protein after protein A affinity chromatography. In some embodiments, protein purity is detected by SEC-HPLC techniques. In some preferred embodiments, the IL-2 mutein products of the invention can be more than 70%, or 80%, or 90% pure after one-step protein A affinity chromatography purification.

In some embodiments, relative to wild-type IL-2 (e.g., the IL-2 shown in SEQ ID NO:26)^WT) The binding affinity of the IL-2 muteins of the invention to the IL-2R α receptor is reduced by at least 5-fold, at least 10-fold, or at least 25-fold, in particular by at least 30-fold, 50-fold or more than 100-fold. In a preferred embodiment, the muteins of the invention do not bind to IL-2 receptor alpha. Binding affinity the equilibrium dissociation constant (K) of an IL-2 mutein of the invention, e.g., an IL-2 mutein of the invention fused to an Fc fragment, and the receptor IL-2R α receptor can be determined by Biofilm Layer Interference (BLI) techniques_D) To be determined. In some embodiments, the monovalent binding affinity of an IL-2 mutein (e.g., in the form of an Fc fusion) to the receptors IL-2R α or IL-2R β is determined by BLI techniques.

In some embodiments, relative to wild-type IL-2 (e.g., the IL-2 shown in SEQ ID NO:26)^WT) The binding affinity of the IL-2 muteins of the invention to the IL-2R β receptor is enhanced at least 5-fold, at least 10-fold, or at least 25-fold, in particular at least 30-fold, 50-fold, or 100-fold, more preferably at least 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or 500-fold or 550-fold or more. Binding affinity the equilibrium dissociation constant (K) of an IL-2 mutein of the invention, e.g., an IL-2 mutein of the invention fused to an Fc fragment, to the receptor IL-2R β receptor can be determined by Biofilm Layer Interference (BLI) techniques_D) To be determined. In one embodiment, the IL-2 muteins of the invention, in the form of IL-2-Fc fusion proteins, have a monovalent binding affinity K for the receptor IL-2R beta receptor in a BLI assay (e.g., the BLI assay described in the examples)_DValues of less than 10.0E-07M, such as 8.0E-07M to 1.0E-07M, such as 4.0E-07M,3.0E-07M, 2.0E-07M,1.0E-07M, more preferably less than 10.0E-08M, such as less than 9.0E-10M.

In one embodiment, the IL-2 muteins of the invention result in reduced IL-2 mediated CD25 relative to wild-type IL-2⁺Cell activation and proliferation. In one embodiment, CD25⁺CellsIs CD25⁺CD8 ⁺T cells. In another embodiment, CD25⁺The cells are Treg cells. In one embodiment, the IL-2 mutein is detected in CD25 in a STAT5 phosphorylation assay⁺Activation of STAT5 phosphorylation signal in cells to identify IL-2 muteins activating CD25⁺The capacity of the cell. For example, as described in the examples herein, the half maximal effective concentration (EC50) can be determined by flow cytometry analysis of STAT5 phosphorylation in cells.

In one embodiment, the IL-2 muteins of the invention result in a maintained or enhanced IL-2 mediated CD25 relative to wild-type IL-2^-Effector cell activation and proliferation. In one embodiment, CD25^-The cell is CD8⁺Effector T cells or NK cells. In one embodiment, the IL-2 mutein is detected in CD25 in a STAT5 phosphorylation assay^-Identification of IL-2 muteins activating CD25 by activating EC50 values of STAT5 phosphorylation signal in cells^-The capacity of the cell. In one embodiment, the IL-2 muteins of the invention activate CD25 relative to the wild-type IL-2 protein (e.g., human IL-2 of SEQ ID NO:26), as determined in a STAT5 phosphorylation assay⁺The capacity of the cell is increased at least 1-fold, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold.

In one embodiment, the IL-2 muteins of the invention remove or reduce IL-2 vs CD25 relative to wild-type IL-2⁺A preference for preferential activation of cells. In one embodiment, CD25⁺The cell is CD25⁺CD8 ⁺T cells. In another embodiment, CD25⁺The cells are Treg cells. In one embodiment, IL-2 muteins are detected in CD25, respectively, in a STAT5 phosphorylation assay^-Cellular neutralization is at CD25⁺Identification of IL-2 muteins activating CD25 by activating EC50 values of STAT5 phosphorylation signal in cells^-The capacity of the cell. For example, by calculation at CD25^-And CD25⁺Determination of the ratio of EC50 values of the phosphorylation signal of activated STAT5 on T cells for IL-2 mutein vs CD25⁺The activation of the cells is biased. Preferably, the mutein pair CD25 is compared to the wild-type protein⁺Is reduced by at least 10 times, preferably at least 100 times, 150 times, or 200 times.

Muteins of the invention

Glycosylated muteins

In one aspect, the invention provides IL-2 muteins comprising a mutated glycosylation motif at the IL-2 and IL-2Ra binding interface.

As is known in the art, polypeptides are typically glycosylated via an N-linkage or an O-linkage. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine (N X S) and asparagine-X-threonine (N X T) are N-linked glycosylation motifs, wherein X is any amino acid except proline. The presence of any of these tripeptide sequences in a polypeptide will result in potential glycosylation sites. The addition of N-linked glycosylation sites to a protein (e.g., IL-2) can be conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences. For example, N-linked glycosylation sites can be added by altering the codons for a single amino acid. For example, codons encoding N-X-z (where z is any amino acid) can be altered to encode N-X-T (or N-X-S), or codons encoding y-X-T/S can be altered to encode N-X-T/S. Alternatively, codons encoding two amino acids can be changed simultaneously to introduce N-linked glycosylation sites (e.g., codons for y-X-z can be changed to encode N-X-T/S).

Herein, glycosylation motifs, which occur in IL-2 proteins due to introduced mutations, can be described as mutant glycosylation motifs. For example, the mutant glycosylation motif K35N-L36-T37 is an N-linked glycosylation motif formed by the substitution of lysine at position 35 for asparagine, while residues 36 and 37 remain unchanged. In a preferred embodiment of the invention, the mutated glycosylation motif introduced is an N-linked glycosylation motif, N-X-S/T, wherein X is any amino acid except proline. In some embodiments, for example, X may be the same amino acid as the amino acid at the corresponding position in wild-type IL-2, or a conservatively substituted residue thereof.

In some embodiments, the present invention provides IL-2 glycosylation muteins comprising at least one mutation that introduces one or more glycosylation motifs N-X-S/T at an amino acid position selected from the group consisting of:

35N-36X-37T/S; 38N-39X-40T/S; 41N-42X-43T/S; 43N-44X-45T/S; 45N-46X-47T/S; 62N-63X-64T/S; 68N-69X-70T/S; 72N-73X-74T/S; 74N-75X-76T/S, wherein X is any amino acid except proline, preferably X is the same amino acid as the amino acid at the corresponding position in wild type IL-2, or a conservatively substituted residue thereof; wherein the amino acid positions are numbered according to SEQ ID NO: 26. In some embodiments, the number of N-linked glycosylation sites introduced can be more than one, such as two glycosylation sites. Different glycosylation sites can confer different properties on IL-2, e.g., some glycosylation sites can confer improved expression and/or purification properties, and some glycosylation sites can improve IL-2 receptor selectivity. In still other embodiments, the invention of the mutant protein can also contain through mutation introduced glycosylation motif, and at least 1-30 different amino acid residues from wild type IL-2, such as 1-20,1-15, 1-10, or 1-5 different amino acid residues. These different residues may be conservative substitutions, or may be other mutations that confer other improved properties to IL-2.

Glycosylation mutations that improve drug-forming properties

In some embodiments, the mutant glycosylation motif improves the pharmaceutical properties of the IL-2 protein, particularly facilitates expression and/or purification of the IL-2 protein.

In one embodiment, the mutated glycosylation motif that improves drug-forming properties is selected from: 35N-36X-37T/S; 38N-39X-40T/S; and 74N-75X-76T/S. In a preferred embodiment, the mutant glycosylation motif is selected from: (i) K35N-L36-T37; (ii) R38N-M39-L40S; and (iii) Q74N-S75-K76T. In a more preferred embodiment, the mutant glycosylation motif is K35N-L36-T37.

Thus, in some embodiments, the invention provides IL-2 muteins comprising a mutant glycosylation motif selected from the group consisting of: 35N-36X-37T/S; 38N-39X-40T/S; and 74N-75X-76T/S, and the mutant protein has improved druggability. In one embodiment, the mutation may facilitate expression and/or purification of the mutein when expressed in mammalian cells, preferably as an Fc fusion protein. In yet another embodiment, the mutation may promote the stability of IL-2, e.g., having a reduced tendency to form aggregates during production compared to wild-type IL-2 when expressed as an Fc fusion protein. For example, after expression and one-step protein a affinity purification, the mutein may have a higher purity than the wild-type protein. In a preferred embodiment, the mutein comprises, compared to wild-type IL-2, a mutated glycosylation motif selected from: (i) K35N-L36-T37; (ii) R38N-M39-L40S; (iii) Q74N-S75-K76T; more preferably the mutein comprises the mutated glycosylation motif K35N-L36-T37.

In some embodiments, the mutant glycosylation motif is introduced into the IL-2 protein by mutation of K35N. In some embodiments, the present invention provides IL-2 muteins having an amino acid sequence that differs from SEQ ID NO: the mature region of the wild type IL-2 protein listed in one of 26,29 or 30 has a mature region of at least 90% identity and has the amino acid residue T37 and the mutation K35N.

In some embodiments, the mutant glycosylation motif is introduced into the IL-2 protein by a pair-wise mutation selected from R38N/L40S or Q74N/K76T. In some embodiments, the present invention provides IL-2 muteins having an amino acid sequence that differs from SEQ ID NO: the mature region of the wild type IL-2 protein listed in one of 26,29 or 30 has a mature region of at least 90% identity and has a pair-wise mutation selected from R38N/L40S or Q74N/K76T.

In some embodiments, the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 31, 32 and 38. In yet another preferred embodiment, the mutein comprises the amino acid sequence SEQ ID NO 31, 32 and 38.

Glycosylation mutations that reduce IL-2R alpha binding

In some embodiments, the mutant glycosylation motif improves receptor selectivity of the IL-2 protein, particularly reduces IL-2 binding to IL-2R α.

In one embodiment, the mutant glycosylation motif that reduces IL-2 binding to IL-2R α is selected from the group consisting of: 41N-42X-43T/S; 43N-44X-45T/S; 45N-46X-47T/S; 68N-69X-70T/S; 72N-73X-74T/S, preferably a glycosylation motif 43N-44X-45T/S, wherein the amino acid positions are numbered according to SEQ ID NO: 26. In a preferred embodiment, the mutant glycosylation motif that reduces IL-2 binding to IL-2R α is selected from the group consisting of: (i) T41N-F42-K43S; (ii) K43N-F44-Y45T; (iii) Y45N-M46-P47S; (iv) E68N-V69-L70S; (v) L72N-A73-Q74T; more preferably, K43N-F44-Y45T.

Thus, in some embodiments, the invention provides an IL-2 mutein comprising a mutated glycosylation motif, as compared to wild-type IL-2, wherein the mutein comprises one or more mutated glycosylation motifs selected from the group consisting of: 41N-42X-43T/S; 43N-44X-45T/S; 45N-46X-47T/S; 68N-69X-70T/S; 72N-73X-74T/S, preferably a glycosylation motif 43N-44X-45T/S, wherein the amino acid positions are numbered according to SEQ ID NO:26, and wherein the mutein has reduced or eliminated IL-2Ra binding compared to wild-type IL-2.

In yet another embodiment, the invention provides an IL-2 mutein comprising a mutated glycosylation motif compared to wild-type IL-2, wherein the mutein comprises one or more mutated glycosylation motifs selected from the group consisting of: (i) T41N-F42-K43S; (ii) K43N-F44-Y45T; (iii) Y45N-M46-P47S; (iv) E68N-V69-L70S; (v) L72N-A73-Q74T; more preferably, the mutein comprises the mutated glycosylation motif K43N-F44-Y45T.

In some embodiments, the mutant glycosylation motif is identified by a sequence selected from T41N/K43S; K43N/Y45T; Y45N/P47S; E68N/L70S; and L72N/Q74T, into the IL-2 protein. In some embodiments, the present invention provides IL-2 muteins having an amino acid sequence that differs from SEQ ID NO:26,29 or 30, and having a mature region at least 85% or 90% identical to the mature region of a wild-type IL-2 protein as set forth in one of T41N/K43S; K43N/Y45T; Y45N/P47S; E68N/L70S; and L72N/Q74T, preferably with the paired mutations K43N/Y45T. In some embodiments, the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: 33,34,35,37, and 39 SEQ ID NOs.

In some embodiments, in addition to the above-described mutant glycosylation motif that reduces binding of IL-2 to IL-2R α, an IL-2 mutein can further comprise: (i) selected from 35N-36X-37T/S; 38N-39X-40T/S; and a mutated glycosylation motif of 74N-75X-76T/S; and/or (ii) the mutation K35Q. The muteins have reduced or abolished IL-2Ra binding compared to wild-type IL-2 and improved expression and/or purification properties (e.g., when expressed in mammalian cells as an Fc fusion protein). In some preferred embodiments, the present invention provides IL-2 muteins having an amino acid sequence that differs from SEQ ID NO:26,29 or 30, and having a mature region at least 85% or 90% identical to the mature region of a wild-type IL-2 protein as set forth in one of T41N/K43S; K43N/Y45T; Y45N/P47S; E68N/L70S; and L72N/Q74T, and has a mutation selected from the group consisting of K35N; R38N/L40S; Q74N/K76T; or a mutation of K35Q. In some embodiments, the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS 45-47.

B 'C' ring chimeric muteins and truncated muteins

In one aspect, the invention provides B 'C' loop chimeric IL-2 muteins and truncated IL-2 muteins formed by introducing mutations into the B 'C' loop region of IL-2. IL-2 proteins belong to a short-chain type I cytokine family member with a four alpha helix bundle (A, B, C, D) structure. As used herein, "B 'C' loop region" or "B 'C' loop sequence" are used interchangeably and refer to the linker sequence between the B and C helices of the IL-2 protein. In one embodiment, the linker sequence is a sequence linking the residue at position 72 and the residue at position 84 in the IL-2 polypeptide according to the numbering of SEQ ID NO: 26. In the wild-type proteins of SEQ ID NO 26,29 and 30, the linker sequence comprises 11 amino acids A73-R83.

In some embodiments, the introduced mutation results in a mutein comprising a shortened B 'C' loop region (i.e., a shortened length of the linker sequence between amino acid residues aa72 and aa 84) compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO:26), preferably said shortened loop region has a length of less than 10, 9,8, 7, 6 or 5 amino acids, and preferably 7 amino acids, wherein the amino acid residues are numbered according to SEQ ID NO: 26.

In some embodiments, the IL-2 muteins of the present invention are B 'C' loop chimeric muteins. The muteins comprise substitutions of the aa73 to aa83 sequences, for example short B 'C' loop sequences from other four-helix short chain cytokine family members, relative to wild-type IL-2. Short B 'C' loops suitable for replacement of wild-type IL-2 can be identified by crystal structure superpose from other four-helix short chain cytokine IL family members, such as IL-15, IL-4, IL-21, or IL family members from non-human species (e.g., mice). In a preferred embodiment, the sequence used for substitution is the B 'C' loop sequence from interleukin IL-15, especially human IL-15. Preferably, the sequence of residues 73-83 in wild type IL-2 is replaced with the sequence SGDASIH.

In some embodiments, the IL-2 muteins of the present invention are B 'C' loop truncation muteins. The muteins comprise a truncation of the aa73 to aa83 sequence, e.g. 1, 2, 3 or 4 amino acids from the C-terminus, relative to the wild-type IL-2. Preferably, the truncated loop region (i.e. the linker sequence between position 72 and position 84) has the sequence a (Q/G) S (K/a) N (F/I) H, preferably said truncated loop region has the sequence AQSKNFH or AGSKNFH.

In one embodiment, the stability of the B 'C' loop, and thus the stability of IL-2 and/or affinity for IL-2R β, may be increased by substitution or truncation of the B 'C' loop. Thus, in one embodiment, the invention provides IL-2 muteins with increased stability and/or increased IL-2 rbeta binding affinity relative to wild-type IL-2, said muteins comprising the aforementioned B 'C' loop chimeric mutation or B 'C' loop truncation mutation, in particular the alternative loop sequence sgihdas or the truncated loop sequence AQSKNFH or AGSKNFH, between position 72 and position 84.

In one embodiment, the chimeric B 'C' ring mutation or truncated B 'C' ring mutation not only confers increased IL-2R β binding, but may also facilitate the expression and/or purification of IL-2 protein, particularly in mammalian cell expression systems. Thus, in one embodiment, the invention provides IL-2 muteins with enhanced IL-2R β binding and/or improved expression and/or purification properties relative to wild-type IL-2. The IL-2 muteins comprise the aforementioned B 'C' loop chimeric mutations or B 'C' loop truncation mutations, in particular the alternative loop sequence SGDASIH or the truncated loop sequence AQSKNFH or AGSKNFH, between position 72 and position 84.

In some preferred embodiments, the present invention provides IL-2 muteins having an amino acid sequence that differs from SEQ ID NO:26,29 or 30, and comprises a linker sequence between amino acid positions 72 and 84 selected from the group consisting of: SGDASIH; AQSKNFH; AGSKNFH; AQSANFH; and AQSANIH. In some embodiments, the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: 40-44, preferably 40-42, more preferably 40 or 41.

Combinatorial muteins

In one aspect, the invention provides IL-2 muteins comprising combinatorial mutations. In one embodiment, the glycosylation mutations introduced into the IL-2 and IL-R α binding interface can be combined with each other, as well as with the B 'C' loop mutation, preferably with the B 'C' loop mutation described herein. In another embodiment, the B 'C' ring mutations of the invention may also be combined with glycosylation mutations introduced at the binding interface of IL-2 and IL-R α, preferably with glycosylation mutations described herein. In a preferred embodiment, by combining the B 'C' loop mutation with a glycosylation mutation introduced into the IL-2 and IL-R α binding interface, improved properties selected from two or all three of the following may be provided: (i) reduced (or eliminated) IL-2R alpha binding; (ii) enhanced IL-2ra binding, and (ii) improved expression levels and purification.

Thus, in one embodiment, the present invention provides an IL-2 mutein, wherein the mutein comprises the combined mutations: (i) selected from 41N-42X-43T/S; 43N-44X-45T/S; 45N-46X-47T/S; 68N-69X-70T/S; a mutated glycosylation motif of 72N-73X-74T/S; and (ii) a shortened B 'C' loop region sequence selected from the group consisting of SGDASIH and A (Q/G) S (K/A) N (F/I) H between amino acid positions aa72 to aa84, wherein the amino acid positions are numbered according to SEQ ID NO: 26.

In some preferred embodiments, the present invention provides IL-2 muteins having an amino acid sequence that differs from SEQ ID NO:26,29 or 30, and comprises a linker sequence between amino acid positions 72 and 84 selected from the group consisting of: SGDASIH; AQSKNFH; AGSKNFH; AQSANFH; and AQSANIH; and having paired mutations selected from: T41N/K43S; K43N/Y45T; Y45N/P47S; E68N/L70S; and L72N/Q74T. In some preferred embodiments, the present invention provides IL-2 muteins having an amino acid sequence that differs from SEQ ID NO:26,29 or 30, and comprises a linker sequence between amino acid positions 72 and 84 selected from the group consisting of: SGDASIH; AQSKNFH; or AGSKNFH; and has paired mutations: K43N/Y45T. In some embodiments, the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: 48, 49 or 50, preferably 48 or 49. In some embodiments, the mutein consists of SEQ ID NO: 48. 49 or 50.

In some embodiments, the combinatorial mutation results in IL-2 with reduced preferential stimulation of CD25⁺Preference for p-STATA5 signaling in T cells with enhanced stimulation of CD25^-The ability to signal in T cells. Thus, in one embodiment, the invention also provides an IL-2 mutein comprising the combined mutations:

(i) a mutated glycosylation motif K43N-F44-Y45T at amino acid positions 43-45 and a substitution sequence SGDASIH between amino acid positions aa72 to aa 84; or

(ii) A mutated glycosylation motif K43N-F44-Y45T at amino acid positions 43 to 45 and a truncated sequence AQSKNFH between amino acid positions aa72 to aa84,

and the muteins have a reduced preferential stimulation of CD25 compared to wild-type IL-2⁺Preference for p-STATA5 signaling in T cells with enhanced stimulation of CD25^-The ability to signal in T cells. Preferably, the mutein comprises the sequence of SEQ ID NO 48 or 49, or a sequence having at least 95%, 96%, or more identity thereto. More preferably, the mutein consists of the sequence of SEQ ID NO 48 or 49.

Other mutations

In addition to the above region and position mutations, the IL-2 mutant protein can also have one or more mutations in other regions or positions, as long as it retains the IL-2 mutant protein of the invention of the above one or more beneficial properties. For example, the IL-2 muteins of the present invention may also comprise a substitution at position 125, such as C125S, C125A, C125T, or C125V, to provide additional advantages, such as improved expression or homogeneity or stability (see, e.g., U.S. patent No. 4,518,584). The skilled artisan knows how to determine additional mutations that can be incorporated into the IL-2 muteins of the present invention.

The sequence differences between the IL-2 mutein and the wild-type protein can be expressed in terms of sequence identity or in terms of the number of amino acids that differ between the two. In one embodiment, the IL-2 mutein has at least 85%, 86%, 87%, 88%, 89% identity, preferably more than 90% identity, preferably 95%, but preferably not more than 97%, more preferably not more than 96% identity to the wild type protein. In another embodiment, in addition to the glycosylation mutations or B 'C' loop mutations or a combination of mutations of both described above, the IL-2 mutein may have no more than 15, such as 1-10, or 1-5 mutations, from the wild-type protein. In one embodiment, the remaining mutations may be conservative substitutions.

2. Fusion proteins and immunoconjugates

The invention also provides fusion proteins comprising the IL-2 muteins of the invention. In a preferred embodiment, the IL-muteins of the invention are fused to another polypeptide which confers improved pharmacokinetic properties, such as albumin, more preferably an antibody Fc fragment. In one embodiment, the Fc fragment comprises a mutation that reduces or removes effector function, for example, a L234A/L235A mutation or a L234A/L235E/G237A that reduces binding to fey receptors. Preferably, the Fc-containing fusion protein has an increased serum half-life. In a preferred embodiment, the Fc-containing fusion protein also has reduced effector functions mediated by the Fc region, such as reduced or eliminated ADCC or ADCP or CDC effector functions.

In one embodiment, the invention also provides IL-2 mutein-Fc fusion proteins wherein the Fc fragment comprises effector functions, such as ADCC. Wild-type IL-2 may deplete Treg cells via Fc-mediated immune effector function, particularly mediated by binding Fc γ R, by fusion to Fc, as reported in the literature (Rodrigo Vazzez-Lombardi et al, supra), thereby improving tumor therapy. Therefore, the IL-2 muteins of the present invention having improved production properties such as expression and/or purification, fused with an Fc fragment retaining immune effector functions, also fall under the contemplation of the present invention. In one embodiment, the fusion protein comprises the mutation K35N or K35Q or the pair of mutations R38N/L40S or Q74N/K76T. In other embodiments, the fusion protein comprises the alternative sequence SGDASIH or the truncated sequence A (Q/G) S (K/A) N (F/I) H between amino acid positions aa72 to aa 84. In one embodiment, the fusion protein comprises greater than 90% to 99% identity to the amino acid sequence of SEQ ID NO 7,8,14, 20-22. In another embodiment, the fusion protein comprises NO more than 0-10 or 0-5 amino acid mutations from the amino acid sequence of SEQ ID NO 12.

In some embodiments, the IL-2 mutein is fused to the Fc via a linker. In some embodiments, the linker may be selected to increase Fc fusion protein to CD25^-Activation of T cells. In one embodiment, the linker is GSGS, more preferably 2x (G4S).

In some embodiments, the Fc fusion protein comprises at least 85%, at least 95%, or at least 96% identity to an amino acid sequence selected from the group consisting of seq id nos: 3-13 and 16-25 of SEQ ID NO. In some embodiments, the Fc fusion protein consists of the sequences of SEQ ID NOS 3-13 and 16-25.

The invention also provides an immunoconjugate comprising the IL2 mutein of the invention and an antigen binding molecule. Preferably, the antigen binding molecule is an immunoglobulin molecule, in particular an IgG molecule, or an antibody or antibody fragment, in particular a Fab molecule and an scFv molecule. In some embodiments, the antigen binding molecule specifically binds to an antigen present on a tumor cell or in the tumor environment, for example an antigen selected from the group consisting of: fibroblast Activation Protein (FAP), the a1 Domain of tenascin C (TNC a1), the a2 Domain of tenascin C (TNC a2), the ectodomain B of fibronectin (Extra Domain B, EDB), carcinoembryonic antigen (CEA), and melanoma-associated chondroitin sulfate proteoglycan (MCSP). Thus, the immunoconjugates of the invention can target tumor cells or the tumor environment after administration to a subject, thereby providing further therapeutic benefits, such as the feasibility of treatment at lower doses and the consequent low side effects; enhanced anti-tumor effects, and the like.

In the fusion proteins and immunoconjugates of the invention, the IL-2 muteins of the invention can be linked, directly or through a linker, to another molecule or antigen-binding molecule, and in some embodiments comprise a proteolytic cleavage site between the two.

3. Polynucleotides, vectors and hosts

The invention provides nucleic acids encoding any of the IL-2 muteins or fusions or conjugates above. The polynucleotide sequence encoding the mutein of the present invention can be generated by de novo solid phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding wild-type IL-2 using methods well known in the art. In addition, the polynucleotides and nucleic acids of the invention may comprise a segment encoding a secretion signal peptide operably linked to a segment encoding a mutein of the invention, such that secretory expression of the mutein of the invention may be directed.

The invention also provides vectors comprising the nucleic acids of the invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or Yeast Artificial Chromosomes (YACs). In a preferred embodiment, the expression vector of the present invention is the pYDO _017 expression vector.

The invention also provides a host cell comprising said nucleic acid or said vector. Suitable host cells for replicating and supporting the expression of the mutant IL-2 protein or fusion or immunoconjugate are well known in the art. Such cells can be transfected or transduced with a particular expression vector and large numbers of vector-containing cells can be grown for use in seeding large-scale fermentors to obtain sufficient quantities of IL-2 mutants or fusions or immunoconjugates for clinical use. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell or 293 cell). For example, polypeptides can be produced in bacteria, particularly when glycosylation is not required. After expression, the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding polypeptides, including fungal and yeast strains in which the glycosylation pathway has been "humanized", resulting in the production of polypeptides having a partially or fully human glycosylation pattern. See Gerngross, NatBiotech22,1409-1414(2004) and Li et al, NatBiotech24,210-215 (2006). Examples of mammalian host cell lines that may be used are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293T cells, as described, for example, in Graham et al, JGenVirol36,59(1977)), Lorentz mouse kidney cells (BHK), mouse Sertoli (Sertoli) cells (TM4 cells, as described, for example, in Mather, biol Reprod23,243-251(1980)), monkey kidney cells (CV1), African Green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT060562), TRI cells (as described, for example, in Mather et al, AnnaadlsN.Y.AcSci 383,44-68(1982), C5 cells, and MR 4 cells. Other mammalian host cell lines that may be used include Chinese Hamster Ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al, ProcNatlAcad Sci USA77,4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp 2/0. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) cell, or a lymphocyte (e.g., Y0, NS0, Sp20 cell).

4. Preparation method

In a further aspect, the invention provides a method of preparing an IL-2 mutein or fusion or conjugate of the invention, wherein said method comprises culturing a host cell comprising a nucleic acid encoding said protein or fusion or conjugate, as provided above, under conditions suitable for expression of the IL-2 mutein or fusion or conjugate, and optionally recovering said protein or fusion or conjugate from said host cell (or host cell culture medium).

5. Assay method

The IL-2 muteins provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.

In one aspect, the IL-2 muteins of the present invention can be tested for binding activity to the IL-2 receptor. For example, binding to human IL-2R α or β protein can be determined by methods known in the art, such as ELISA, Western blotting, and the like, or by the exemplary methods disclosed in the examples herein. For example, the flow cytometry assay can be used, wherein cells transfected to express a mutein on the cell surface, such as yeast display cells, are reacted with a labeled (e.g., biotin labeled) IL-2R α or β protein. Alternatively, the mutant protein and receptor binding, including binding kinetics (e.g. K)_DValues), can be determined in a Biofilm Layer Interference (BLI) assay using recombinant mutein-Fc fusions. In some embodiments, the BLI assay as described in the examples is used.

In yet another aspect, signaling and/or immune activation effects that occur downstream of receptor binding can be measured. To indirectly measure the ability of the IL-2 mutein to bind to the IL-2 receptor.

Thus, in some embodiments, assays for identifying mutant IL-2 proteins having biological activity are provided. Biological activities may include, for example, the ability to induce proliferation of T and/or NK cells and/or Treg cells having an IL-2 receptor, the ability to induce IL-2 signaling in T and/or NK cells and/or Treg cells having an IL-2 receptor, a reduced ability to induce apoptosis in T cells, the ability to induce tumor regression and/or improve survival, and reduced in vivo toxicity properties, such as reduced vascular permeability. The invention also provides mutant IL-2 proteins having such biological activities in vivo and/or in vitro.

Various methods are known in the art for determining the biological activity of IL-2. For example, a suitable assay for testing the ability of an IL-2 mutein of the invention to stimulate IFN- γ production by NK cells may comprise the following steps: cultured NK cells were incubated with the mutant IL-2 proteins or fusion or immunoconjugates of the invention and subsequently the IFN- γ concentration in the culture medium was measured by ELISA. IL-2 signaling induces several signaling pathways and involves JAK (Janus kinase) and STAT (activator of signal transduction and transcription) signaling molecules.

The interaction of IL-2 with the beta and gamma subunits of the receptor results in phosphorylation of the receptor as well as JAK1 and JAK3 (which bind to the beta and gamma subunits, respectively). STAT5 then binds to phosphorylated receptors and phosphorylates itself on very important tyrosine residues. This results in dissociation of STAT5 from the receptor, dimerization of STAT5, and translocation of STAT5 dimers to the nucleus, where they promote transcription of target genes. Thus, the ability of a mutant IL-2 polypeptide to induce signaling via the IL-2 receptor can be assessed, for example, by measuring phosphorylation of STAT 5. Details of this method have been disclosed in the examples. For example, PBMCs may be treated with a mutant IL-2 polypeptide or fusion or immunoconjugate of the invention and the level of phosphorylated STAT5 determined by flow cytometry.

In addition, T cell or NK cell proliferation in response to IL-2 can be measured by incubating T cells or NK cells isolated from blood with a mutant IL-2 polypeptide or immunoconjugate of the invention, followed by determination of ATP content in lysates of the treated cells. Prior to treatment, T cells may be pre-stimulated with phytohemagglutinin (PHA-M). This assay allows sensitive quantification of the number of viable cells, and a number of suitable alternative assays (e.g. [3H ] -thymidine incorporation assay, cell titration GloATP assay, AlamarBlue assay, WST-1 assay, MTT assay) are also known in the art.

Furthermore, the effect of mutated IL-2 on tumor growth and survival can be assessed in a variety of animal tumor models known in the art. For example, xenografts of human cancer cell lines can be implanted into immunodeficient mice and treated with mutant IL-2 polypeptides or fusions or immunoconjugates of the invention. Toxicity of the mutant IL-2 polypeptides, fusions and immunoconjugates of the invention in vivo can be determined based on mortality, life-time observations (visible symptoms of adverse effects, e.g., behavior, body weight, body temperature), and clinical and anatomical pathology (e.g., measurement of blood chemistry values and/or histopathological analysis). For example, vascular permeability induced by treatment with IL-2 can be examined in a pretreated vascular permeability animal model with a vascular leakage reporter molecule. Preferably, the vascular leak reporter is large enough to reveal permeability of the wild-type form of IL-2 for pretreatment.

Furthermore, for IL-2 glycosylated muteins of the present invention, the presence, absence or degree of glycosylation can also be determined by any method known to those skilled in the art, including semi-quantitative measurements of Molecular Weight (MW) shift, as observed by Western blotting or from Coomassie-stained SDS-PAGE gels, while quantitative measurements can include the use of mass spectrometer techniques and observation of the MW shift corresponding to the addition of asparagine-linked glycosylation, or mass shifts associated with the removal of asparagine-linked glycosylation by enzymes such as peptide-N-glycoglycase F (PNGase-F; Sigma Aldrich, St.Louis, Mo.).

6. Screening method

In a further aspect, the present invention provides a method for obtaining an IL-2 mutein with improved properties.

In one embodiment, the present invention provides a method for obtaining an IL-2 mutein comprising the steps of:

-artificially engineering one or more (e.g. two or three) glycosylation motifs N-X-S/T (X may be any amino acid except P (proline)) at the binding interface of IL-2 and IL-2Ra, preferably introducing a glycosylation motif in a region selected from the group consisting of IL-2: aa35-40, aa41-47, aa62-64, aa68-74, aa 74-76;

-allowing expression of the engineered IL-2 mutein, e.g. in the form of an Fc fusion (e.g. FcLALA fusion), in mammalian cells (e.g. HEK293 or CHO cells). For the design of sites for the introduction of glycosylation motifs, N-glycosylation prediction tools can be used to select sites that can be mutated to promote potential N-linked glycosylation, for example by identifying residues that can be mutated to form standard N-x-T/S glycosylation sites (where N is asparagine and x is any amino acid except proline). In addition, a structure-based approach can be used to identify IL-2 as being a distance from IL-2R α

And the side chain is exposed to amino acids in solution as mutationsAre candidate amino acids for asparagine. In some preferred embodiments, the introduced glycosylation motif mutation is selected from: K35N-L36-T37; R38N-M39-L40S; T41N-F42-K43S; K43N-F44-Y45T; Y45N-M46-P47S; E62N-L63-K64T; E68N-V69-L70S; L72N-A73-Q74T; Q74N-S75-K76T.

In another aspect, the present invention provides a method for obtaining an IL-2 mutein comprising the steps of:

-introducing deletions and/or substitutions in the B ' C loop region (aa73-83) of IL-2 to form a shortened loop region, preferably to replace it with the B ' C loop sequence of other four-helix short chain cytokine family members, such as IL15, to form a B ' C loop chimera, or truncating the B ' C loop of IL-2 to form a B ' C loop truncate, preferably the shortened loop region has a length of less than 10, 9,8, and preferably equal to 7 amino acids; preferably 1, 2, 3 or 4 amino acids from the C-terminus of the loop region; preferably the shortened loop region has the sequence A (Q/G) S (K/A) N (F/I) H, or SGDASIH;

-allowing expression of the engineered IL-2 mutein, e.g. in the form of an Fc fusion (e.g. FcLALA fusion), in mammalian cells (e.g. HEK293 or CHO cells).

In one embodiment, the method further comprises: following protein expression and purification, IL2 muteins with improved drug-properties (e.g., expression levels and/or product stability and/or homogeneity, e.g., one-step Fc affinity chromatography purity) were identified. In a preferred embodiment, glycosylation motif mutations are introduced in the aa35-40 or aa74-76 regions of IL-2 to improve the druggability of the mutein. Preferably, the introduced glycosylation motif mutation is selected from: K35N-L36-T37; R38N-M39-L40S; and Q74N-S75-K76T. In another preferred embodiment, the druggability of the mutein is improved by replacing the B 'C' loop with a shortened loop, for example the loop sequence of IL15 or by truncating the B 'C' loop. Preferably, the shortened loop sequence is selected from: a (Q/G) S (K/A) NFH, or SGDASIH. In a further preferred embodiment, the method comprises, in addition to the glycosylation mutation, the introduction of other point mutations, such as K35Q, to improve the drugability of the mutein. As the technicians in this field is clear, these mutations and endowing other improved properties of the mutation combination, to obtain with multiple improved properties of IL-2 mutant protein.

In one embodiment, the method further comprises: IL-2 muteins were identified which exhibit a reduced (preferably abolished) IL-2Ra binding capacity relative to wild-type IL-2. In one embodiment, the IL-2 mutant protein and IL-2Ra binding capacity by determination of affinity KD values, for example by biomembrane thin layer interference technology determination. In yet another embodiment, binding capacity is determined by assaying IL-2 mutein for CD25⁺The efficacy of activation of T cells. In one embodiment, the IL-2 mutein exhibits reduced CD25 relative to wild-type IL-2⁺T cell activation efficacy, for example, as determined by measuring activation of p-STAT5 signal in cells. Mutations are preferably introduced into the region of IL-2: aa41-47 or aa68-70 or aa72-74 to form potential N-linked glycosylation sites, and then to examine whether the mutation results in reduced or eliminated binding of IL-2 to IL-2R α. Preferably, the introduced glycosylation motif mutation is selected from: T41N-F42-K43S; K43N-F44-Y45T; Y45N-M46-P47S; E68N-V69-L70S; L72N-A73-Q74T. As the technicians in this field is clear, can be these glycosylation mutations and other improved properties of the mutation combination, to obtain multiple improved properties of IL-2 mutant protein.

In one embodiment, the method further comprises identifying an IL-2 mutein that exhibits enhanced IL-2R β binding relative to wild-type IL-2. In one embodiment, the IL-2 mutant protein and IL-2R beta binding capacity is determined by measuring affinity KD value, for example by biological membrane thin layer interference technology determination. In yet another embodiment, binding capacity is determined by assaying IL-2 mutein for CD25^-The efficacy of activation of T cells. In one embodiment, the IL-2 mutant protein, relative to wild type IL-2, shows enhanced CD25^-T cell activation efficacy, for example, as determined by measuring activation of p-STAT5 signal in cells. In a preferred embodiment, the B 'C' loop is replaced by a shortened loop, such as the loop sequence of IL15 or by truncation of the B 'C' loop, to enhance binding to IL-2R β. Preferably, shrinkingThe short loop sequence is selected from: a (Q/G) S (K/A) NFH, or SGDASIH. As the technicians in this field is clear, can be these glycosylation mutations and other improved properties of the mutation combination, to obtain multiple improved properties of IL-2 mutant protein.

In a further embodiment, the method comprises the combined introduction of the above described prodrug-improving mutations, mutations that reduce IL2Ra binding, and/or mutations that enhance IL2R β binding, and/or combinations of mutations that confer other improved properties, to obtain IL-2 muteins with multiple improved properties. In a preferred embodiment, the glycosylation mutations are introduced in combination, for example in the regions aa41-47 and aa68-74, with truncation and/or substitution mutations that shorten the length of the B 'C' loop region. In a preferred embodiment, the method comprises identifying an IL-2 mutein that exhibits reduced IL-2Ra binding and enhanced IL-2R β binding relative to wild-type IL-2, optionally identifying an IL-2 mutein that also has improved druggability (e.g., improved expression and/or purity, and/or product stability and/or homogeneity).

In some embodiments, the parent wild-type IL-2 protein used as a template for the mutation is preferably at least 85%, or at least 90% or 95% identical to SEQ ID NO. 26, more preferably a human-derived IL-2 protein.

7. Pharmaceutical composition and pharmaceutical preparation

The invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising an IL-2 mutein or a fusion or immunoconjugate thereof and compositions comprising a polynucleotide encoding an IL-2 mutein or a fusion or immunoconjugate thereof. These compositions may also optionally contain suitable pharmaceutical excipients such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as are known in the art.

Pharmaceutical carriers suitable for use in the present invention may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. For the use of excipients and their use, see also "Handbook of pharmaceutical excipients", fifth edition, r.c. rowe, p.j.seskey and s.c. owen, pharmaceutical press, London, Chicago. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

Pharmaceutical formulations comprising the present invention may be prepared by mixing an IL-2 mutein, fusion or immunoconjugate of the invention of the desired purity with one or more optional Pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16 th edition, Osol, a. eds. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution. Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulation including histidine-acetate buffer. In addition, sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

The pharmaceutical compositions or formulations of the present invention may also contain one or more other active ingredients that are required for the particular indication being treated, preferably those active ingredients that have complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other anti-cancer active ingredients, such as chemotherapeutic agents, PD-1 axis binding antagonists (e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies). The active ingredients are suitably present in combination in an amount effective for the intended use.

Thus, in one embodiment, the composition further comprises a second therapeutic agent. For example, the second therapeutic agent can be an immune checkpoint inhibitor. For example, the second therapeutic agent may be selected from the group including, but not limited to: for example, anti-CTLA-4 antibodies, anti-CD 47 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CD 40 antibodies, anti-OX 40 (also known as CD134, TNFRSF4, ACT35 and/or TXGP1L) antibodies, anti-LAG-3 antibodies, anti-CD 73 antibodies, anti-CD 137 antibodies, anti-CD 27 antibodies, anti-CSF-1R antibodies, TLR agonists, or one or more small molecule antagonists of IDO or TGF β. Preferably, the second therapeutic agent is a PD-1 antagonist, in particular an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3, an anti-CD 47. In addition to the immunotherapeutic agent, the second therapeutic agent may also be another radiotherapeutic or chemotherapeutic agent.

8. Combination product

In one aspect, the invention also provides a combination product comprising a mutein of the invention or a fusion or immunoconjugate thereof, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective active agents, etc.). The combination product of the invention may be used in the method of treatment of the invention.

In some embodiments, the invention provides a combination product wherein the additional therapeutic agent is, for example, a therapeutic agent such as an antibody effective to stimulate an immune response, thereby further enhancing, stimulating, or up-regulating the immune response in the subject. In some embodiments, the other antibody is, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody or an anti-LAG-3 antibody or an anti-CTLA-4 antibody or an anti-TIM-3 antibody.

In some embodiments, the combination product is for use in the prevention or treatment of a tumor. In some embodiments, the tumor is a cancer, e.g., a gastrointestinal cancer, e.g., gastric, rectal, colon, colorectal, etc.; or skin cancer, such as melanoma; or renal cell carcinoma, bladder cancer, non-small cell lung cancer, etc. In some embodiments, the combination product is used to prevent or treat an infection, such as a bacterial infection, a viral infection, a fungal infection, a protozoan infection, and the like.

9. Methods of treatment and uses

Herein, the terms "individual" or "subject" are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the subject is a human.

As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in the individual undergoing treatment. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis.

In one aspect, the invention provides a method of stimulating the immune system of a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-2 mutein or fusion or immunoconjugate of the invention. IL-2 muteins of the invention against CD25^-CD122 ⁺Effector cells (cytotoxic CD 8)⁺T cells and NK cells) with high activity and selectivity and with reduced CD25⁺Stimulation of Treg cells. Thus, the IL-2 muteins of the present invention can be used at low doses to stimulate the immune system of a subject.

Thus, in some embodiments, the invention relates to a method of enhancing an immune response of a body in a subject, the method comprising administering to the subject an effective amount of any of the IL-2 muteins described herein, or a fusion or immunoconjugate thereof. In some embodiments, the IL-2 muteins or fusions or immunoconjugates thereof of the present invention are administered to a subject bearing a tumor, stimulating an anti-tumor immune response. In other embodiments, an antibody or antigen-binding portion thereof of the invention is administered to a subject harboring an infection to stimulate an anti-infective immune response. In one embodiment the IL-2 muteins of the present invention may be used in combination with a Treg depleting antibody (e.g., Fc γ R mediated Treg depletion) to further reduce the immunosuppressive effects caused by tregs. In one embodiment, the IL-2 muteins of the present invention can be administered in combination with an immune checkpoint inhibitor, e.g., to enhance cancer immunotherapy effects, e.g., in combination with anti-PD-1 and anti-CTLA-4.

In another aspect, the invention relates to a method of treating a disease, such as tumors and cancers and infections, in a subject, the method comprising administering to the subject an effective amount of any of the IL-2 muteins described herein, or a fusion or immunoconjugate thereof.

The cancer may be in an early, intermediate or advanced stage or a metastatic cancer. In some embodiments, the tumor or tumor cell may be selected from colorectal tumors, ovarian tumors, pancreatic tumors, lung tumors, liver tumors, breast tumors, kidney tumors, prostate tumors, gastrointestinal tumors, melanoma, cervical tumors, bladder tumors, glioblastoma, and head and neck tumors. In some embodiments, the cancer may be selected from colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, and head and neck cancer. In some embodiments, the tumor is melanoma, renal cell carcinoma, colorectal cancer, bladder cancer, non-small cell lung cancer.

In another aspect, the invention relates to a method of treating an infectious disease, e.g., a chronic infection, in a subject, the method comprising administering to the subject an effective amount of any of the IL-2 muteins or fragments thereof described herein, or an immunoconjugate, multispecific antibody, or pharmaceutical composition comprising the antibody or fragment. In one embodiment, the infection is a viral infection.

In some embodiments, in addition to the IL-2 muteins or fusions or conjugates thereof of the present invention, the methods of the present invention further comprise co-administering to the subject one or more therapies (e.g., therapeutic modalities and/or other therapeutic agents). In some embodiments, the treatment modality includes surgical treatment and/or radiation therapy. In some embodiments, the methods of the invention further comprise administering at least one additional immunostimulatory antibody, e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CD 43 antibody, and/or an anti-CTLA-4 antibody, which may be, e.g., fully human, chimeric, or humanized.

In some embodiments, the anti-PD-1 antibody is selected from the group consisting of: IBI308 (Xindilizumab, WO2017/025016A1), MDX-1106(nivolumab, OPDIVO), Merck 3475(MK-3475, pembrolizumab, KEYTRUDA) and CT-011 (Pidilizumab). In some embodiments, the anti-PD-1 antibody is MDX-1106. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registry number 946414-94-4). In further embodiments, the IL-2 mutein or fragment thereof alone or in combination with a PD-1 antagonist can also be administered in combination with one or more other therapies, e.g., treatment modalities and/or other therapeutic agents. In some embodiments, the treatment modality includes surgery (e.g., tumor resection); radiation therapy (e.g., external particle beam therapy, which involves three-dimensional conformal radiation therapy in which an irradiation region is designed), localized irradiation (e.g., irradiation directed at a preselected target or organ), focused irradiation, or the like.

In some embodiments, provided herein are methods of treating a disease (e.g., a tumor) comprising administering to a subject a mutein described herein and a CTLA-4 antagonist antibody. The anti-CTLA-4 antibody can be, for example, an antibody selected from the group consisting of:

(ipilimumab or antibody 10D1, described in PCT publication No. WO 01/14424), tremelimumab (formerly ticilimumab, CP-675,206), and anti-CTLA-4 antibodies described in the following publications: WO 98/42752; WO 00/37504; U.S. patent nos. 6,207,156; hurwitz et al (1998) Proc. Natl. Acad. Sci. USA 95(17) 10067-10071; camacho et al (2004) J.Clin.Oncology 22(145) Abstract No.2505 (antibody CP-675206); and Mokyr et al (1998) Cancer Res.58: 5301-5304.

In some embodiments, provided herein are methods of treating a disease (e.g., a tumor) comprising administering to a subject an anti-mutein and an anti-LAG-3 antagonist antibody described herein. The anti-LAG 3 antibody may be, for example, an antibody selected from the group consisting of: antibodies 25F7,26H10,25E3,8B7,11F2, or 17E5, or antibodies comprising the CDRs or variable regions of these antibodies, described in U.S. patent application nos. US2011/0150892 and WO 2014/008218; BMS-986016; IMP731 described in US 2011/007023.

In some embodiments, the IL-2 muteins of the present invention can be administered in combination with a chemotherapeutic or chemotherapeutic agent. In some embodiments, the IL-2 muteins of the present invention can be administered in combination with radiation therapy or a radiotherapeutic agent. In some embodiments, the IL-2 muteins of the present invention can be administered in combination with a targeted therapy or targeted therapeutic. In some embodiments, the IL-2 muteins of the present invention can be administered in combination with an immunotherapy or immunotherapeutic agent, such as a monoclonal antibody.

The muteins of the invention (as well as the pharmaceutical compositions comprising them or fusions or immunoconjugates thereof, and optionally additional therapeutic agents) can be administered by any suitable method, including parenteral, intrapulmonary and intranasal administration, and, if desired for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, for example intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, a single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

For the prevention or treatment of disease, the invention of the mutant protein suitable dosage (when alone or with one or more other therapeutic agent combination use) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether used for the purpose of prevention or treatment, previous treatment, patient clinical history and the antibody response, and the attending physician's judgment. The antibody is suitably administered to the patient as a single treatment or over a series of treatments.

In a further aspect, the invention also provides the use of an IL-2 mutein, composition, immunoconjugate, fusion of the invention in the manufacture of a medicament for use in the aforementioned methods (e.g. for treatment).

The following examples are described to aid in the understanding of the present invention. The examples are not intended to, and should not be construed as, limiting the scope of the invention in any way.

Example 1 design of Interleukin-2 mutants

● design of interleukin-2 glycosylated protein

According to the crystal structure (PDB:1Z92) (figure 1A) of Interleukin-2 (Interleukin-2, IL-2 for short) and an alpha receptor CD25 (IL-2R alpha for short) in a PDB database, an N-X-S/T motif (X can be any amino acid except P (proline)) is artificially modified at the binding interface of IL-2 and IL-2Ra by site-directed mutation of amino acids, so that the IL-2 forms a polysaccharide chain on the surface of the IL-2 through post-translational modification of cells in the expression process of HEK293 or CHO cells, and the binding of the IL-2 and the IL-2R is blocked (shown in the structural diagram of figure 1B).

IL-2 glycosylation site design: finding the distance from IL-2Ra in IL-2

And the side chain is exposed to the amino acid in the solution, the amino acid is mutated into asparagine, and the following third amino acid is mutated into serine or threonine to form an N-X-S/T motif (X can be any amino acid except P), which is shown in Table 1.

TABLE 1 mutation sites of IL-2 glycosylated proteins

Name (R)	Mutant amino acids and N-X-S/T motifs
L007(IL-2 ^glycan1)	K35N-L36-T37
L 008(IL-2 ^glycan2)	R38N-M39-L40S
L 009(IL-2 ^glycan3)	E68N-V69-L70S
L 010(IL-2 ^glycan4)	Y45N-M46-P47S
L 011(IL-2 ^glycan5)	K43N-F44-Y45T
L 012(IL-2 ^glycan6)	E62N-L63-K64T
L 013(IL-2 ^glycan7)	L72N-A73-Q74T
L 014(IL-2 ^glycan8)	Q74N-S75-K76T
L 015(IL-2 ^glycan9)	T41N-F42-K43S

● design of IL-2B 'C' loop chimeras and truncators

B 'C' loop: the linker sequence of B helix and C helix of IL-2 (FIG. 2A), comprises 11 amino acids A73-R83.

Comparing the crystal structures of IL-2 monomer (PDB:1M47) and complex (PDB: 2ERJ), we found that the B 'C' loop is absent from the crystal structure of IL-2 monomer, since it is very active in solution and cannot form a relatively stable conformation.

The stability of the B 'C' loop is increased by performing genetic engineering transformation on the B 'C' loop, so that the stability of the IL-2 and the affinity with the IL-2R are increased. We therefore compared the crystal structure of human IL15 (PDB:2Z3Q) and found that its B 'C' loop is shorter and stable (FIG. 2. B). Therefore, we designed one chimeric IL-2 molecule (L017) and 4 truncated molecules (L057 to L060) (see Table 2).

TABLE 2 IL-2B 'C' Loop optimized sequences

Name (R)	B 'C' loop sequence
L 001(IL-2 ^WT)	AQSKNFHLRPR
L 017(IL-2 ^hyb15BCL)	SGDASIH
L 057(IL-2 ^truncate1)	AQSKNFH
L 058(IL-2 ^truncate2)	AGSKNFH
L 059(IL-2 ^truncate3)	AQSANFH
L 060(IL-2 ^truncate4)	AQSANIH

Example 2: expression purification of IL-2 mutant-Fc fusion proteins and IL-2 receptors

Construction of expression plasmids

Wild type IL-2(uniprot: P60568, aa21-153, C125S, IL-2 for short^WT) And IL-2 mutants of IL-2^3X(R38D,K43E,E61R)，IL-2 ^glycansAnd B 'C' loop chimera and truncation, which are connected with Fc (L234A, L235A, FcLALA for short, SEQ ID NO:28) of human IgG1 through a GSGS connecting sequence and constructed on a vector of pTT5 to express the following proteins:

name of protein	Structure of the product	SEQ ID NOs
Y001	IL-2 ^WT-GSGS-FcLALA	SEQ ID NO:1
Y002	IL-2 ^.3X-GSGS-FcLALA	SEQ ID NO:2

Y007	IL-2 ^.glycan1-GSGS-FcLALA	SEQ ID NO:3
Y008	IL-2 ^.glycan2-GSGS-FcLALA	SEQ ID NO:4
Y009	IL-2 ^.glycan3-GSGS-FcLALA	SEQ ID NO:5
Y010	IL-2 ^.glycan4-GSGS-FcLALA	SEQ ID NO:6
Y011	IL-2 ^.glycan5-GSGS-FcLALA	SEQ ID NO:7
Y012	IL-2 ^.glycan6-GSGS-FcLALA	SEQ ID NO:8
Y013	IL-2 ^.glycan7-GSGS-FcLALA	SEQ ID NO:9
Y014	IL-2 ^.glycan8-GSGS-FcLALA	SEQ ID NO:10
Y015	IL-2 ^.glycan9-GSGS-FcLALA	SEQ ID NO:11
Y017	IL-2 ^hyb15BCL-GSGS-FcLALA	SEQ ID NO:12
Y057	IL-2 ^truncate1-GSGS-FcLALA	SEQ ID NO:20
Y058	IL-2 ^truncate2-GSGS-FcLALA	SEQ ID NO:21
Y059	IL-2 ^truncate3-GSGS-FcLALA	SEQ ID NO:22
Y060	IL-2 ^truncate4-GSGS-FcLALA	SEQ ID NO:23

IL-2 ^WT,IL-2 ^3XAnd L011 (IL-2)^glycan5) Was ligated to FcLALA via 2 GGGGS and constructed onto a vector of pcdna3.1 to express the following proteins:

name of protein	Structure of the product	SEQ ID NOs
Y038	IL-2 ^.glycan5-2*(G4S)-FcLALA	SEQ ID NO:13
Y040	IL-2 ^.3X-2*(G4S)-FcLALA	SEQ ID NO:14
Y045	IL-2 ^WT-2*(G4S)-FcLALA	SEQ ID NO:15

At L011 (IL-2)^glycan5) On the basis, 1 glycosylation site is added or a K35Q mutation site is added (the K35Q mutation is designed based on the mutant protein Y007 and the protein 3D structure), and is connected with FcLALA through a GSGS connecting sequence and is constructed on a pTT5 vector; to express the following proteins:

name of protein	Structure of the product	SEQ ID NOs
Y048	IL-2 ^{glycan5。glycan8}-GSGS-FcLALA	SEQ ID NO:16
Y049	IL-2 ^{glycan5。glycan1}-GSGS-FcLALA	SEQ ID NO:17
Y050	IL-2 ^{glycan5。K35Q}-GSGS-FcLALA	SEQ ID NO:18

The B 'C' loop chimera (L017) and the truncation (L057/058) were combined with glycosylated IL-2(L011), linked to FcLALA through 2 GGGGS, and constructed onto a vector of pCDNA3.1 to express the following proteins:

name of protein	Structure of the product	SEQ ID NOs
Y056	IL-2 ^{.glycan5.15BCL}-2*(G4S)-FcLALA	SEQ ID NO:19
Y081	IL-2 ^{.glycan5.truncate1}-2*(G4S)-FcLALA	SEQ ID NO:24
Y082	IL-2 ^{.glycan5.truncate2}-2*(G4S)-FcLALA	SEQ ID NO:25

The specific sequence information of the protein molecules is shown in a sequence table.

Wild-type IL-2 for the construction of the above molecules^WT26, having a C125S mutation at position 125 of the sequence to avoid formation of disulfide-bridged IL-2 dimers. IL-2^3XIs an IL-2 mutant reported in the literature (Rodrigo Vazquez-Lombardi et al, Nature Communications,8:15373, DOI:10.1038/ncomms15373) and IL-2^WTThe same also contains the C125S mutation and contains the mutations R38D, K43E, E61R, the sequence of which is shown in SEQ ID NO: 27. According to the literature, IL-2^3XDoes not bind with IL-2R alpha, and maintains the binding force with IL-2R beta equivalent to that of wild type IL-2.

Expression purification of IL-2 fusion proteins

Expi293 cells (Invitrogen) were passaged according to the desired transfection volume, and the cell density was adjusted to 1.5X 10 the day before transfection⁶Individual cells/ml. Cell density at day of transfection was approximately 3X 10⁶Individual cells/ml. The thus-constructed expression plasmid was added to Opti-MEM medium (Gibco cat # 31985-. Adding appropriate Polyethyleneimine (PEI) (Polysciences, 23966) into the plasmid in the previous step (the mass ratio of the plasmid to the PEI is 1:3), mixing uniformly, and incubating at room temperature for 10min to obtain a DNA/PEI mixture. The DNA/PEI mixture was gently poured into HEK293 cells and mixed well at 37 ℃ with 8% CO₂After 24h of culture under the conditions described above, VPA (Sigma, cat # P4543-100G) was added to a final concentration of 2mM and 2% (v/v) Feed (1G/L Phytone Peptone +1G/L Difco Select Phytone), and the culture was continued for 6 days.

The cell culture broth was centrifuged at 13000rpm for 20min, the supernatant was collected and the supernatant was purified using a pre-packed column Hitrap Mabselect Sure (GE, 11-0034-95). The operation is as follows: the packed column was equilibrated with 5 column volumes of equilibration solution (20mM Tris,150mM NaCl, pH7.2) before purification; passing the collected supernatant through a column, and then cleaning the packed column by using a balance liquid with 10 times of the column volume to remove non-specific binding protein; the packing was washed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5) and the eluate was collected. mu.L of Tris (2M Tris) was added to each 1ml of the eluate, exchanged into PBS buffer (Gibco, cat # 70011-044) using an ultrafiltration concentration tube (MILLIPORE, cat # UFC901096), and the concentration was determined. Mu.g of the purified protein was taken, adjusted to a concentration of 1mg/mL, and the protein purity was determined using a gel filtration chromatography column SW3000(TOSOH cat # 18675).

The glycosylation mutants Y007, Y008 and Y014 obviously improve the expression amount and purity of the protein relative to Y001 after mutating one or two amino acids on the surface. Y048, Y049 and Y050 increase 1 glycosylation site or one K35Q mutation site on the basis of Y011, so that the expression level is increased from 7.77mg/L to more than 50mg/L (Y048 and Y049) or 40mg/L (Y050), the purity is increased from 31.35% to more than 80%, and the drugability of the molecule is remarkably improved.

Compared with Y001, the B 'C' loop chimera (Y017) and the truncation (Y057/058/059) have greatly improved expression amount and one-step affinity chromatography purity.

After combining the B 'C' loop optimized sequence with the glycosylation mutation L011, Y056, Y081 and Y082 were improved in expression and purity compared to the mutein Y011 with L011 (table 3).

TABLE 3 expression and purity of IL-2 mutants in HEK293

● expression and purification of IL-2 receptor

The human IL-2 receptors Uiprot: P01589, aa-217) and (Uiprot: P14784, aa27-240) are linked at the C-terminus of the sequence to an avi tag (a polypeptide: GLNDIFEAQKIEWHE, which can be biotinylated by BirA enzyme catalysis) and 6 histidine tags (HHHHHHHHH), were constructed separately onto pTT5 vector. Plasmid transfection 293F cells (Invitrogen) method with IL-2Fc fusion protein expression method.

The collected medium was centrifuged at 4500rpm for 30min before purification, and the cells were discarded. The supernatant was filtered using a 0.22. mu.l filter. The nickel column used for purification (5ml Histrap excel, GE,17-3712-06) was soaked with 0.1M NaOH for 2h, then washed with 5-10 column volumes of ultra-pure water to remove the alkali solution. The purification column was equilibrated with 5 column volumes of binding buffer (20mM Tris pH 7.4,300mM NaCl) before purification; passing the cell supernatant through the equilibrated column; removing non-specifically bound heteroproteins by passing 10 column volumes of wash buffer (20mM Tris 7.4,300mM NaCl,10mM imidazole) through the column; the target protein was then eluted with 3-5 column volumes of eluent (20mM Tris 7.4,300mM NaCl,100mM Imidazole). The collected proteins were concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-. A100. mu.g sample of the purified protein was taken and the protein purity was determined using a gel filtration chromatography column SW3000(TOSOH cat # 18675) (FIGS. 3 and 4).

Example 3 IL-2 mutant Fc fusion proteins (abbreviation: IL-2)^mutant-FC) affinity assay for its receptor

Measurement of IL-2 of the present invention Using the biofilm thin layer interference (BLI) technique^mutant-equilibrium dissociation constant (KD) for FC binding to human IL-2R α and IL-2R β. The BLI method affinity assay is carried out according to the known methods (Estep, P et al, High throughput solution Based measurement of antibody-antibody affinity and affinity binding. MAbs,2013.5(2): pages 270-8).

Half an hour before the start of the experiment, an appropriate number of AHC (ForteBio, 18-5060) (for positive control detection) sensors were soaked in SD buffer (PBS 1 ×, BSA 0.1%, Tween-200.05%) depending on the number of samples.

Taking 100. mu.l of SD buffer solution and IL-2^mutantFC, IL-2 receptor or separately in 96-well black polystyrene half-well microplates (Greiner, 675076). Sensor locations were selected based on sample location plating. Instrument for measuring the shape of a human bodyThe device setting parameters are as follows: the operation steps are as follows: baseline, Loading-1 nm, Baseline, Association and Dissociation; the run time for each step depends on the sample binding and dissociation speed, 400rpm, and 30 ℃. Analysis of K Using ForteBio analysis software_DThe value is obtained.

TABLE 4a. IL-2^glycanAffinity K of FC for IL-2R_DValue of

Name of protein	Affinity (M) for IL-2R
Y001	1.12E-08
Y002	N.B.
Y007	2.55E-09
Y008	4.23E-08
Y009	N.B.
Y010	N.B.
Y011	N.B.
Y012	9.22E-08
Y013	N.B.
Y014	1.03E-08
Y015	N.B.

Table 4B affinity K of IL-2B 'C' Loop mutants for IL-2R_DValue of

Name of protein	Affinity (M) for IL-2R
Y001	N.B.
Y017	8.87E-08
Y057	2.34E-07
Y058	3.44E-07
Y059	1.46E-07
Y060	7.63E-07

Table 4c affinity of il-2 mutant combinations for receptors K_DValue of

Name of protein	Affinity (M) for IL-2R	Affinity (M) for IL-2R
Y038	N.B	N.B
Y040	N.B	N.B
Y045	5.03E-08	N.B
Y056	N.B	1.20E-07
Y081	N.B	2.60E-07
Y082	N.B	P.F

IL-2 does not bind to the receptor; P.F the bond is very weak and the fitting effect is poor. (ii) a

From the above affinity data it can be seen that: 1) y009, Y010, Y011, Y013 and Y015 can block the binding of IL-2R (Table 4 a); 2) b 'C' loop chimeric molecules and truncated molecules, not only increased the expression level of the molecules, but also increased the affinity of the molecules to IL-2R (Table 4B); 3) IL-2 glycosylation and B 'C' loop engineered combinations Y056 and Y081, and Y045 (IL-2)^WT-2*(G4S)-FcLALA)、Y040(IL-2 ^.3X-2 x (G4S) -FcLALA) and Y038 (IL-2)^.glycan5-2 x (G4S) -FcLALA) increased affinity for IL2R while blocking IL2R binding.

Example 4: IL-2^mutant-FC in vitro functional assay

IL-2 ^WTThe affinity with IL-2R alpha is higher than that of IL-2R beta and IL-2R gamma, the IL-2R alpha on the surface of the cell is preferentially combined, the IL-2R beta gamma is recruited, and downstream p-STAT5 signals are released through the IL-2R beta gamma, so that the proliferation of T cells and NK cells is stimulated. Because the surface of Treg cells has IL-2R alpha, effector T cells and NK cells have no IL-2R alpha, and IL-2 is normally used^WTCan preferentially stimulate the proliferation of Treg cells and down regulate immune response. IL-2^mutantIs not combined with IL-2R alpha, eliminates the preference of preferentially stimulating Treg cell proliferation, and simultaneously stimulates T cells and NK cells to proliferate, so that the number of effector T cells and NK cells is effectively increased, and the anti-tumor effect is improved.

This example was performed by detecting each IL-2^mutantFC vs primary human CD8⁺Activation of T cell p-STAT5 Signal, validation of each mutant on CD25⁺Removal of cell activation bias and screening for CD25^-Mutants with stronger cell activation. The method comprises the following specific steps:

1. and (3) recovering the PBMC cells:

a) PBMC cells were removed from liquid nitrogen (Allcells cat #: PB005F, 100M package), quickly placing in a water bath kettle at 37 ℃, and recovering PBMC cells;

b) cells were added to 10mL of pre-warmed, 5% human AB serum (GemCell cat #: 100-: 07900) X-VIVO15(Lonza cargo number: 04-418Q), washing once by centrifuging at 400G and 25 ℃ for 10 minutes (the subsequent centrifugation is the condition);

c) the cells were resuspended in 20mL medium and incubated overnight at 37 ℃ in a carbon dioxide incubator.

2. Purification of human CD8⁺T cell:

a) absorbing the cell suspension in the step 1, centrifuging and removing supernatant;

b) 1mL of Robosep buffer (STEMCELL cat #: 20104) With 100. mu.L of human AB serum and 100. mu.L of human CD8⁺Resuspend cells in the negative selection antibody cocktail in the T cell purification kit (Invitrogen cat # 11348D);

c) after mixing, incubating for 20 minutes at 4 ℃ and shaking once every 5 minutes;

d) after incubation, 10mL of Robosep buffer was added and washed twice by centrifugation;

e) at the same time, 1mL of magnetic microspheres (human CD 8) were taken⁺T cell purification kit), adding 7mL Robosep buffer solution, placing on a magnetic frame for 1 minute, discarding the supernatant, and prewashing the magnetic microspheres;

f) adding 1mL of Robosep buffer solution into each suspension microsphere and cell respectively, mixing uniformly, and performing rotary incubation for 30 minutes at room temperature;

g) after incubation, 6mL of Robosep buffer solution was added, the mixture was placed on a magnetic frame for 1 minute, and the supernatant was collected;

h) placing the collected liquid on the magnetic frame again for 1 minute, and collecting the supernatant;

i) the supernatant was discarded by centrifugation, resuspended in preheated T medium and adjusted to a density of 1X 10⁶/mL；

j) 1/3 cells are taken to stimulate CD25 expression after the cells are taken, and the rest cells are placed in a carbon dioxide incubator at 37 ℃ for standing overnight for culture.

3. Stimulation of CD8⁺T cells express CD 25:

a) 1/3 taking CD8 purified in step 2⁺T cells, magnetic microspheres (GIBCO cat # 11131D) containing anti-human CD3/CD28 antibody, cells and microspheresThe ratio of the balls is 3: 1;

b) standing in a carbon dioxide incubator at 37 ℃ for three days;

c) adding 10mL of culture medium, and cleaning for 2 times;

d) adding culture medium to adjust cell density to 1 × 10⁶mL, static culture in a carbon dioxide incubator at 37 ℃ for 2 days.

4. Detecting the purity and expression level of the cells:

a) anti-human CD8-PE (Invitrogen cat no: 12-0086-42), anti-human CD25-PE (eBioscience cat No.: 12-0259-42), isotype control antibody (BD cat No.: 556653) detecting CD8 and CD25 of the cells;

b) the cells in step 2 were CD8⁺CD25 ^-T cells, step 3 cells are CD8⁺CD25 ⁺T cells.

5. Detection of each IL-2^mutant-FC vs CD8⁺CD25 ^-EC for T cell activation of p-STAT5 signaling₅₀：

a) Fetching CD8⁺CD25 ^-T cells at 1X 10 per well⁵Cells were plated in 96-well U-bottom plates (Costar cat # CLS3799-50 EA);

b) add 100. mu.L of each IL-2^mutant-FC, commercial IL-2 (R)&D, cargo number: 202-IL-500), IL-2^WT-FC、IL-2 ^3X-FC, maximum concentration starting from 266.7nM with 12 gradients diluted in 4-fold gradients, incubated for 20min in an incubator at 37 ℃;

c) adding 55.5 μ L of 4.2% formaldehyde solution, and fixing at room temperature for 10 min;

d) the supernatant was discarded by centrifugation, and 200. mu.L of ice methanol (Fisher Cat #: a452-4) resuspending the cells, incubating for 30 minutes at 4 ℃ in a refrigerator;

e) the supernatant was discarded by centrifugation, and the supernatant was washed with 200. mu.L of a staining buffer (BD cat #: 554657) 3 washes;

f) 200 μ L of a solution containing anti-p-STAT 5-AlexFlour647(BD cat #: 562076, 1:200 dilution) of membrane rupture/fixation buffer (BD stock No.: 51-2091KZ), and incubating for 3 hours at room temperature in a dark place;

g) washing the cells for three times by using a staining buffer solution, resuspending the cells by using 100 mu L of the staining buffer solution, and detecting the cells by using a flow cytometer;

h) EC for making p-STAT5 signal with IL-2 molecule concentration as abscissa and AlexFlour647 intermediate fluorescence value as ordinate₅₀The results are shown in FIG. 5 and Table 5.

6. Detection of each IL-2^mutant-FC vs CD8⁺CD25 ⁺EC for T cell activation of p-STAT5 signaling₅₀：

a) Fetching CD8⁺CD25 ⁺T cells at 1X 10 per well⁵Laying 96-hole U-bottom culture plates for cells;

b) preparing EC of p-STAT5 signal in the same step b-h as step 5₅₀The results are shown in FIG. 5 and Table 5.

TABLE 5 IL-2 mutant vs CD25^+/-EC for T cell activation of p-STAT5 signaling₅₀And their ratio (donor2)

Donor2	R&D IL2	Y045	Y040	Y056	Y081
CD25 ⁺pSTAT5 EC ₅₀	0.005086	0.0282	4.12	0.06085	2.186
CD25_pSTAT5 EC ₅₀	0.5945	13.17	34.61	2.856	7.203
CD 25-EC 50/CD 25+ EC50 times	116.8895	467.0213	8.4005	46.9351	3.2951

The experimental results show (comparison under the same donor):

1) comparative Y001 (IL-2)^WTGSGS-FcLALA) and Y045 (IL-2)^WT-2*(G4S)-FcLALA)，Y002(IL-2 ^.3XGSGS-FcLALA) and Y040 (IL-2)^.3X-2*(G4S)-FcLALA)，Y011(IL-2 ^.glycan5GSGS-FcLALA) and Y038 (IL-2)^.glycan5-2 x (G4S) -FcLALA) curve positions, it was found that long linker sequences (GGGGSGGGGS) were superior to short linker sequences (GSGS) for CD25^-CD8 ⁺Activation of T cells (fig. 5A).

2) After chimerization with the B 'C' loop of human IL-15, Y017 (IL-2)^hyb15BCLGSGS-FcLALA) vs CD25^-CD8 ⁺Activation of T cells (EC50 value of 0.9902 for Y017) vs Y001 (EC)₅₀Value of 10.69) increased by a factor of 10.79 (fig. 5A); for CD25⁺CD8 ⁺Activation of T cells (EC50 value of 0.0018 for Y017) and Y001 (EC)₅₀Value 0.0020) (fig. 5B).

3) Addition of one N-glycan at the IL-2 interface (Y038), for CD25^-CD8 ⁺Activation of T cells (EC of Y038)₅₀Value 369.0) compared with wild type IL-2(Y045, EC)₅₀Value of 31.73) decreased by 11.63 times, but better than the IL-2 reported in the literature^3X(Y040); after chimerization with the B 'C' loop of human IL-15 on this basis (Y056, EC)₅₀Value 8.571), for CD25^-CD8 ⁺T cell activation was 3.7-fold higher than Y045 and 43.05-fold higher than Y038 (fig. 5C).

4) CD8+ T cells before and after contrast stimulation under the same donor, suggesting that Y056 and Y081 enhance CD25^-CD8 ⁺Activation of T cells with reduced CD25⁺Cell activation bias (fig. 5D.E and table 5).

Sequence listing

Claims

An IL-2 mutein, wherein the mutein comprises at least one mutation compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO:26) introducing one or more glycosylation motifs N-X-S/T at amino acid positions selected from the group consisting of:

35N-36X-37T/S; 38N-39X-40T/S; 41N-42X-43T/S; 43N-44X-45T/S; 45N-46X-47T/S; 62N-63X-64T/S; 68N-69X-70T/S; 72N-73X-74T/S; 74N-75X-76T/S, wherein X is any amino acid except proline, preferably X is the same amino acid as the amino acid at the corresponding position in wild type IL-2, or a conservatively substituted residue thereof;

wherein the amino acid positions are numbered according to SEQ ID NO: 26.
The mutein of claim 1, wherein the mutein comprises one or more mutated glycosylation motifs selected from the group consisting of: 35N-36X-37T/S; 38N-39X-40T/S; and 74N-75X-76T/S, wherein the amino acid positions are numbered according to SEQ ID NO:26,

wherein the mutein has improved expression and/or purity when expressed in mammalian cells, preferably in the form of an Fc fusion protein, compared to wild-type IL-2 (preferably by determining the purity of the mutein after expression and one-step affinity purification).
The mutein of claim 2, wherein the mutein comprises, compared to wild-type IL-2, a mutated glycosylation motif selected from:

(i)K35N-L36-T37；

(ii)R38N-M39-L40S；

(iii) Q74N-S75-K76T; preferably the mutein comprises the mutated glycosylation motif K35N-L36-T37.
The mutein according to claims 2 and 3, wherein the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: 31, 32 and 38 in SEQ ID NO.
The mutein according to claim 1, wherein the mutein comprises one or more mutated glycosylation motifs selected from the group consisting of: 41N-42X-43T/S; 43N-44X-45T/S; 45N-46X-47T/S; 68N-69X-70T/S; 72N-73X-74T/S, preferably a glycosylation motif 43N-44X-45T/S, wherein the amino acid positions are numbered according to SEQ ID NO:26,

wherein the mutein has reduced or eliminated IL-2Ra binding compared to wild-type IL-2.
The mutein of claim 5, wherein the mutein comprises, compared to wild-type IL-2, a mutated glycosylation motif selected from:

(i)T41N-F42-K43S；

(ii)K43N-F44-Y45T；

(iii)Y45N-M46-P47S；

(iv)E68N-V69-L70S；

(v)L72N-A73-Q74T；

preferably, the mutein comprises the mutated glycosylation motif K43N-F44-Y45T.
The mutein of claims 5 to 6, wherein the mutein further comprises:

(i) selected from 35N-36X-37T/S; 38N-39X-40T/S; and a mutated glycosylation motif of 74N-75X-76T/S; and/or

(ii) The mutation K35Q was found to be,

wherein the mutein has reduced or eliminated IL-2Ra binding compared to wild-type IL-2 and has improved expression and purity when expressed in mammalian cells as an Fc fusion protein.
The mutein according to claims 5 to 7, wherein the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: 33,34,35,37, 39, and 45-47 of SEQ ID NO.
An IL-2 mutein, wherein said mutein comprises a shortened B 'C' loop region (i.e. the linking sequence between amino acid residues aa72 and aa 84), preferably said shortened loop region having an amino acid length of less than 10, 9,8, 7, 6, or 5, and preferably 7 amino acids in length, wherein the amino acid residues are numbered according to SEQ ID NO:26, compared to wild type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO: 26).
The mutein according to claim 9, wherein the mutein comprises, relative to the wild-type IL-2

(i) Substitutions of the aa73 to aa83 sequences, for example short B 'C' loop sequences from a member of the four-helix short chain cytokine IL family, such as the B 'C' loop sequence of IL15, preferably a substituted loop region having the sequence SGDASIH; or

(ii) Truncations of the aa73 to aa83 sequence, e.g. 1, 2, 3 or 4 amino acids from the C-terminus; preferably the truncated loop region has the sequence A (Q/G) S (K/A) N (F/I) H, preferably said truncated loop region has the sequence AQSKNFH or AGSKNFH.
The mutein according to claims 8-10, wherein the mutein has enhanced IL-2R β binding, and/or improved expression yield and/or purity relative to wild-type IL-2.
The mutein of claims 8 to 11, wherein the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: 40-44, preferably 40-42, more preferably 40 or 41.
An IL-2 mutein, wherein the mutein comprises, compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO:26), the combined mutations: (i) selected from 41N-42X-43T/S; 43N-44X-45T/S; 45N-46X-47T/S; 68N-69X-70T/S; a mutated glycosylation motif of 72N-73X-74T/S; and (ii) a shortened B 'C' loop region sequence selected from SGDASIH and A (Q/G) S (K/A) N (F/I) H between amino acid positions aa72 to aa84, wherein the amino acid positions are numbered according to SEQ ID NO: 26.
The mutein of claim 13, wherein the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: 48, 49 or 50, preferably 48 or 49.
The mutein according to claims 1 to 14, wherein the mutein has one or more of the following properties compared to wild-type IL-2:

having an eliminated or reduced binding affinity to the IL-2R alpha receptor,

-has enhanced binding affinity to the IL-2R β receptor;

-has reduced binding affinity to a high affinity IL-2R receptor (IL-2R α β γ);

-has increased binding affinity to the intermediate affinity IL-2R receptor (IL-2R β γ);

-reducing activation of CD25+ cells (in particular CD8+ T cells, in particular Treg cells);

-reducing the stimulatory effect on IL-2 mediated signalling in CD25+ cells (in particular CD8+ T cells);

-removing or reducing the bias of IL-2 to preferentially activate CD25+ cells (especially Treg cells);

-reducing the down-regulation of the immune response by IL-2 induced Treg cells;

maintaining or enhancing the activation of CD 25-cells,

-stimulating the proliferation and activation of effector T cells and NK cells;

-enhancing the anti-tumor effect.
The IL-2 mutein of claim 1-15, wherein the mutein has one or more of the following characteristics when expressed in a mammalian cell, such as a HEK293 cell:

-an amount of expression superior to the wild type IL-2 protein;

-stability superior to wild type IL-2 protein;

easy purification to higher protein purity, e.g. higher purity after one-step affinity chromatography purification.
The IL-2 mutein of any one of claims 1-16, wherein the mutein has at least 85%, at least 95%, or at least 96% identity compared to wild-type IL-2.
The IL-2 mutein of any one of claims 1-16, wherein the mutein has a reduced preference for preferentially stimulating p-STATA5 signaling in CD25+ T cells and an enhanced ability to stimulate signaling in CD25-T cells compared to wild-type IL-2,

preferably the mutein comprises a combination of mutations:

(i) a mutated glycosylation motif K43N-F44-Y45T at amino acid positions 43-45 and a substitution sequence SGDASIH between amino acid positions aa72 to aa 84; or

(ii) A mutated glycosylation motif K43N-F44-Y45T at amino acid positions 43 to 45 and a truncated sequence AQSKNFH between amino acid positions aa72 to aa84,

more preferably, the mutein comprises the sequence of SEQ ID NO 48 or 49, or a sequence having at least 95%, 96%, or more identity thereto.
An IL-2 mutein fusion protein comprising the IL2 mutein according to claims 1-18, preferably fused to an Fc antibody fragment, preferably the IL-2 mutein is fused to Fc by a linker, preferably GSGS, more preferably 2x (G4S),

preferably, the fusion protein comprises at least 85%, at least 95%, or at least 96% identity to an amino acid sequence selected from the group consisting of seq id no:3-13 and 16-25 of SEQ ID NO.
An immunoconjugate comprising the IL2 mutein of claims 1-18 and an antigen binding molecule, preferably the antigen binding molecule is an immunoglobulin molecule, in particular an IgG molecule, or an antibody or antibody fragment, in particular a Fab molecule and an scFv molecule.
The immunoconjugate of claim 20, wherein said antigen binding molecule specifically binds to an antigen present on a tumor cell or in a tumor environment, such as an antigen selected from: fibroblast Activation Protein (FAP), the a1 Domain of tenascin C (TNC a1), the a2 Domain of tenascin C (TNC a2), the ectodomain B of fibronectin (Extra Domain B, EDB), carcinoembryonic antigen (CEA), and melanoma-associated chondroitin sulfate proteoglycan (MCSP).
An isolated polynucleotide encoding the IL-2 mutein of claims 1 to 18 or the fusion of claim 19 or the immunoconjugate of claims 20 to 21.
An expression vector comprising the polynucleotide of claim 22.
A host cell comprising the polynucleotide of claim 22 or the vector of claim 23, preferably said host cell is a mammalian cell, in particular a HEK293 cell, and a yeast.
A method of producing an IL-2 mutein or a fusion or immunoconjugate thereof comprising culturing the host cell of claim 24 under conditions suitable for expression of said IL-2 mutein or fusion or conjugate.
A pharmaceutical composition comprising an IL-2 mutein according to claim 1 to 18 or a fusion according to claim 19 or an immunoconjugate according to claim 20 to 21 and a pharmaceutically acceptable carrier.
A method of treating a disease in a subject, the method comprising administering to the subject an IL-2 mutein of claim 1 to 18 or a fusion of claim 19 or an immunoconjugate of claims 20 to 21 or a pharmaceutical composition of claim 26, preferably the disease is cancer.
A method of stimulating the immune system of a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-2 mutein of claims 1 to 18 or a fusion of claim 19 or an immunoconjugate of claims 20 to 21.
A method for obtaining an IL-2 mutein comprising the steps of:

introducing one or more (e.g.two or three) glycosylation motifs N-X-S/T by mutation at the binding interface of IL-2 and IL-2Ra, wherein X can be any amino acid except P (proline), and/or shortening the loop sequence by mutation in the B 'C' loop region of IL-2,

preferably a glycosylation mutation as described in claims 1 to 7 and/or a B 'C' loop sequence mutation as described in claims 9 to 10, more preferably a combinatorial mutation as described in claims 13 or 18;

expression of IL-2 muteins in mammalian cells (e.g. HEK293 or CHO cells), for example in the form of Fc fusions (e.g. FcLALA fusions);

-identifying a mutein having one or more of the following improved properties: (i) expression level and/or stability; (ii) reducing IL2R α binding; (iii) enhanced IL2R β binding.